CAP1-Mediated m6A Modification of RRM2 Suppresses Tumor-Associated M2 Macrophage Polarization and Colorectal Cancer Growth

CAP1-Mediated m6A Modification of RRM2 Suppresses Tumor-Associated M2 Macrophage Polarization and Colorectal Cancer Growth | 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 Research Article CAP1-Mediated m6A Modification of RRM2 Suppresses Tumor-Associated M2 Macrophage Polarization and Colorectal Cancer Growth Tianlin Feng, Ling Lin, Xiaoya Zhou, Li Li, Yao Chen, Wenyi Zheng, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7355735/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jan, 2026 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted 5 You are reading this latest preprint version Abstract Colorectal cancer (CRC) progression is critically regulated by dynamic interactions between tumor cells and tumor-associated macrophages (TAMs), which shape the immuno-suppressive tumor microenvironment. In this study, we identify CAP1 as a novel regulator of this crosstalk through its control of RNA methylation-dependent M2 macrophage polarization. Clinical analysis reveals significant CAP1 downregulation in CRC tissues compared to adjacent normal mucosa, with its expression positively correlating with patient survival outcomes. While CAP1 knockdown did not show cell-autonomous effects on CRC proliferation in vitro, it dramatically enhanced tumor growth in immuno-competent mouse models. Further mechanistic studies uncover that CAP1 deficiency in tumor cells triggers a ALKBH5-mediated increase in m⁶A RNA methylation, specifically enhancing the translation and secretion of RRM2. This tumor-derived RRM2 potently drives M2 polarization of TAMs, creating a pro-tumorigenic niche that facilitated the proliferation of CRC cells in turn. Moreover, the RRM2 inhibitor shows considerable efficacy in treating tumors with low expression of CAP1. Collectively, this study provides new insights into how tumor cells regulate immune responses through post-transcriptional modification and suggests potential therapeutic strategies targeting the CAP1-RRM2 axis in CAP1-deficient CRCs. colorectal cancer CAP1 RRM2 m6A Macrophage Polarization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Colorectal cancer (CRC) is one of the most common malignant tumors, with the second highest number of new diagnoses in women and the third highest in men, and causing approximately 90,000 deaths annually worldwide [ 1 – 3 ] . Advancements in medical science have led to significant improvements in the clinical management of colorectal cancer (CRC). Traditional treatments, including surgical resection, chemotherapy and radiotherapy, combined with emerging therapies such as targeted therapy and immunotherapy have collectively enhanced patient outcomes. Nevertheless, these treatments still face many challenges including high recurrence rates, severe adverse reactions, inadequate drug specificity, and the development of resistance. Consequently, reveal the molecular mechanisms of CRC pathogenesis and identify novel therapeutic targets are essential to further improve clinical efficacy and patient prognosis. Cyclase-associated protein (CAP) was first identified in yeast as an adenylyl cyclase-associated binding protein with two mammalian homologs, CAP1 and CAP2. CAP2 is a highly structured multifunctional protein with six structural domains [ 4 – 7 ] . Canonically, it regulates the actin cytoskeleton and Ras regulation of adenylyl cyclase [ 8 ] . However, recent studies shown that CAP1 plays an important role in the development of tumors, in breast cancer patients, the expression level of CAP1 was significantly positively correlated with the survival time of patients [ 9 ] . CAP1 plays critical role in linking the major second messenger cAMP to the activation of adherent Rap1 in colon cancer cells, which may also regulate the proliferation of other cell types [ 10 ] . as a receptor of human resistin, CAP1 is involved in immune cell-mediated inflammation by upregulating the concentration of cyclic AMP (cAMP) concentration, protein kinase A (PKA) activity, and NF-κB-related transcription of inflammatory cytokines [ 11 , 12 ] . However, the mechanism of CAP1 in the immune microenvironment of CRC has not been thoroughly studied. The interactions between cancer cells and tumor microenvironment (TME) are dynamic and reciprocal. Tumor microenvironment includes all non-cancerous host cells, including fibroblasts, endothelial cells, neurons, adipocytes, adaptive and innate immune cells, as well as its non-cellular components, including the extracellular matrix, and soluble products such as chemokines, cytokines, growth factors, and extracellular vesicles [ 13 – 15 ] . Tumor-associated macrophages (TAM) are abundantly present in tumor microenvironment of most cancer types and the interaction between cancer cells and TAM shapes the tumor immune landscape are usually associated with clinical prognosis in cancer patients [ 16 , 17 ] . Macrophages in the tumor immune microenvironment can be divided into two subtypes: M1-type macrophages mainly play a role in tumor suppression, promotion of inflammation, and immunoreactivity, whereas M2-type macrophages play a role in tissue repair, immune escape, and promotion of tumorigenesis. The proportion of macrophages in tumor microcircuits is highly correlated with tumorigenesis, severity, and prognosis [ 18 – 21 ] . Ribonucleotide reductase M2 (RRM2) catalyzes the formation of deoxyribonucleotides from ribonucleotides [ 22 ] . studies have shown that RRM2 plays an important role in tumor cell growth [ 23 ] , drug resistance and immunotherapy [ 24 , 25 ] . Xiong et al found that RRM2 stabilizes ANXA1 and activates the AKT pathway independent of its ribonucleotide reductase activity, promoting sunitinib resistance in RCC. Moreover, RRM2 affects the anti-tumor effect of PD-1 antibody by regulating the expression of PD-L1 in tumor cells. At the same time, the expression of RRM2 is positively correlated with the infiltration of M2 macrophages in tumor tissues, and further promotes the growth of tumors [ 24 ] . inhibition of RRM2 promotes macrophage M1 polarization and inhibits M2 polarization in lung adenocarcinoma [ 26 ] . More importantly, RRM2 inhibitor Osalmid significantly enhances Radiosensitivity of Esophageal Cancer and suppressed tumor growth [ 27 ] . In this study, we have demonstrated that CAP1 is lowly expressed in CRC and inhibits tumor growth and regulating the CRC growth through modulating the tumor immune microenvironment, Mechanistically, CAP1 regulates the expression and secretion of RRM2 protein through ALKBH5-mediated m6A modification, thereby affecting the polarization of M2 macrophages and inhibiting the growth of tumors, Moreover the combination of RRM2 inhibitor osalmid and low expression of CAP1 significantly inhibits the growth of CRC. The role of the CAP1/RRM2 axis has potential clinical applications and provides a new strategy for the comprehensive treatment of CRC(Fig. 6 ). Materials and Methods Cell culture MC38 and CT26 were purchased from the Cell Resource Center, Institute of Basic Medical Sciences, CAMS/PUMC. All cell lines were authenticated using short tandem repeat (STR) profiling and routinely tested for mycoplasma contamination. Primary macrophages and cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and maintained at 37°C in a humidified atmosphere of 5% CO 2 . Patient Tissue Specimens With the consent of the patients, a total of 11 cases of resected specimens from colorectal cancer patients were collected for this study from February 2025 to May 2025. colorectal cancer specimens were compared with the paired adjacent cancer tissues from the same patient. and the study was approved by Ethics Committee of Fourth People's Hospital of Chong qing (Chongqing Emergency Medical Center) (Approval Number: 2025 (54)). Mice All animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of Chongqing University Central Hospital (Approval Number: 2412004), Six-week-old male C57BL/6 mice and BALB/c Nude mice were purchased from Jiangsu Huachuang sino Pharma Tech(Jiangsu china) and housed in pathogen-free ventilated cages under controlled conditions (12 h light/dark cycle, 22 ± 1°C, 50 ± 5% humidity) with ad libitum access to autoclaved food and sterile water. Only healthy mice, free from infectious diseases and parasites, were selected, and mice with health issues, abnormal weight changes were excluded. The sample size was chosen based on the literature and our previous experience. mice randomly assigned to different groups. For the peritoneal metastasis model, mice were intraperitoneally injected with 4 × 10 6 MC38 or CT26 cells suspended in 100 µL PBS. After 14 days, mice were euthanized and peritoneal tumor nodules were excised and weighed. For the subcutaneous tumor model, mice received a subcutaneous injection of 4 × 10^6 MC38 cells in 100 µL PBS, and tumor-bearing mice were sacrificed on day 14 or 18 for tumor collection and weight measurement. All the mice were euthanized by cervical dislocation after isoflurane anesthesia, and the tumors were collected. Establishment of CAP1-silenced MC-38 or CT-26 cell lines Lentiviral vectors for CAP1 knockdown were generated by Genechem. (Shanghai, China). The following shRNA sequences were used: shCAP1-1 (5'-GGCTTACATCAAGGAGTTT-3'), shCAP1-2 (5'-TCTACCTTTCTGCTCTCTTAA-3'), and negative control shRNA (shNC, 5'-TTCTCCGAACGTGTCACGT-3'). The lentivirus were employed to infect MC38 and CT26 cells at a multiplicity of infection (MOI) of 10. After 48 hours incubation, the culture medium was supplemented with 5 µg/mL puromycin to selectively screen for successfully transduced cells. The knockdown efficiency of the target gene CAP1 was subsequently assessed using RT-qPCR and western blot. ELISA The medium supernatant levels of RRM2 were quantified using commercial ELISA kits (mlbio, Shanghai, China) following the manufacturer's protocol. Briefly, microplates were equilibrated at room temperature for 1 min before use. Standard solutions (50 µL/well) were added to designated wells in duplicate, while 50 µL of each sample was loaded into sample wells. Blank wells received nothing. After adding 100 µL of horseradish peroxidase (HRP)-conjugated detection antibody to all wells except blanks, plates were sealed and incubated at 37°C for 60 min. Following incubation, wells were emptied and washed five times with 350 µL washing buffer (1 min per wash). Then, 50 µL of chromogenic substrates A and B were added to each well, followed by incubation for 15 min at 37°C. The reaction was terminated by adding 50 µL stop solution, and absorbance was measured at 450 nm within 15 min using a microplate reader. A standard curve was generated by plotting the mean optical density values against corresponding standard concentrations, and sample concentrations were calculated using the linear regression equation derived from the standard curve. Western blotting Cells were washed three times with ice-cold PBS, followed by lysis in RIPA buffer supplemented with protease and phosphatase inhibitors (MCE, Shanghai, China). Protein concentrations were determined using the BCA Protein Assay Kit, with absorbance measured at 562 nm using a microplate reader (Epizyme biotech, Shanghai, China). Equal amounts of protein (30 µg) were separated by 10% SDS-PAGE and transferred onto PVDF membranes at appropriate voltages based on target protein molecular weights. Membranes were blocked with 5% skim milk in TBST at room temperature, followed by incubation with primary antibodies (Supplementary Table 1) at 4°C overnight. After three 5-min TBST washes, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Following another three TBST washes, protein bands were visualized using enhanced chemiluminescence substrate (Ncmbio, Suzhou, china) and imaged using a chemiluminescence detection system (Vilber, Paris, France). RNA m6A Dotblot Assays Total RNA was extracted using the Pure RNA Isolation Kit (Ncmbio, Suzhou, china) and diluted into a gradient concentration of 400 ng/µL, 200 ng/µL, 100 ng/µL and 50 ng/µL. Samples (400 ng, 200 ng, 100 ng, and 50 ng) that degenerated under 95° for 3 min were deposited on an Nitrocellulose membrane (ABclonal, Wuhan, China). Then, the membrane was crosslinked by ultraviolet rays for 5 min and washed with PBST. Subsequently, the membrane was blocked in 5% non-fat milk at room temperature for 1 hour and incubated with primary m6A antibody (ABclonal, Wuhan, China) overnight at 4 C. Dot blots were visualized after incubation with HRP-conjugated secondary antibody. After photography the membrane was stained with 0.02% methylene blue (Solarbio Beijing China) and washed with ddH 2 O until the background turned white, and then photographed. Quantitative real-time PCR Total RNA was extracted using the Pure RNA Isolation Kit (Ncmbio, Suzhou, china) following the manufacturer's instructions. RNA concentration and purity were determined by NanoDrop One (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was synthesized from 1 ug total RNA using the ABScript III RT Master Mix for Qpcr with gDNA Remover according to the manufacturer's protocol (ABclonal, Wuhan, China). Quantitative real-time PCR (qPCR) was performed using 2X Universal SYBR Green Fast qPCR Mix (ABclonal, Wuhan, China) with each reaction containing 1 µL of cDNA template in a final volume of 20 µL. Quantitative PCR was performed using a PCR system(Bio-Rad, Hercules, CA, USA) and relative expression was calculated by the 2 (−∆∆Ct) method. The primers were listed in Supplementary Table 2. Immunohistochemistry Tumor tissue specimens were collected in accordance with protocols approved by the Scientific Research Committee of The Fourth People's Hospital of Chongqing, following ethical guidelines for human subject research. All (CRC) samples were obtained from treatment-naive patients undergoing surgical resection, with proper anonymization procedures. Tissues were fixed in 10% neutral buffered formalin and embedded in paraffin using standard protocols. Immunohistochemistry was performed according to manufacturer's instructions (Zsbio, Beijing, China). Briefly, 4-µm thick sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Antigen retrieval was performed by microwave heating in citrate buffer (pH 6.0) for 10 min. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide at room temperature for 10 min. Non-specific binding was blocked with 10% normal goat serum for 30 min at 37°C. Sections were then incubated with primary antibody against CAP1 overnight at 4°C. After washing with PBS, sections were incubated with HRP-conjugated secondary antibody for 20 min at 37°C, Signals were visualized with diaminobenzidine followed by hematoxylin counterstaining, dehydrated in ethanol, cleared in xylene, and mounted. Isolation of fat macrophages from epididymal adipose tissue Mice were sacrificed via cervical vertebrae dislocation after isoflurane anesthesia. The epididymal adipose tissue was collected, cut and added into DMEM medium supplemented with 1 mg/ml collagenase IV (Sangon Biotech, China), 3% fetal bovine serum and 2% penicillin streptomycin, and digested at 37° for 1h. The digestive solution was filtered through 200 mesh sieve and centrifuged at 500g for 10min. After the removal of red blood cells, the cell pellet was re-suspended in PBS to produce single cell suspension. Flow cytometry Isolated primary single cell or RAW264.7 cells were incubated with TruStain FcX™ PLUS for 10 minutes on ice to block Fc receptors. Subsequently, the cells were resuspended in PBS containing Zombie dyes solution and incubated at room temperature in dark for 20 minutes. For cell-surface staining, primary antibodies (mouse CD45, mouse F4/80, and mouse CD11c) were applied at their predetermined optimal concentrations, and the cells were incubated on ice for 20 minutes in the dark. Next, cells were incubated with the cyto-fast™ Fix/Perm solution at room temperature and protected from light for 20 minutes. Following this, mouse CD206 staining was performed at room temperature for 20 minutes, Finally, the stained cells were resuspended in PBS and analyzed by flow cytometry (Thermo Fisher Scientific, Waltham, MA, USA). M1-like cells macrophages were defined as CD45 + F4/80 + CD206 − CD11c + . M2-like macrophages were characterized as CD45 + F4/80 + CD11c − CD206 + defined. (all reagents and antibodies used in this study were sourced from BioLegend (San Diego, CA), antibodies used were listed in Supplementary Table 1. Co-culture tumor cells with macrophage RAW264.7 Transwell co-culture system (Corning, Glendale, AZ, USA): CAP1 knockdowns tumor cells were seeded in the upper chamber, while RAW264.7 macrophages were cultured in the lower chamber. After 48 h of co-culture, RAW264. 7 cells were collected for immunophenotyping analysis using flow Cytometry. Conditioned medium induce Macrophage polarization Obtain Conditioned medium (CM): CAP1 knockdowns tumor cells were cultured in T-175 flasks with complete medium until reaching 80% confluence. The medium was replaced with RPMI-1640 containing 1% fetal bovine serum, and cells were incubated for 48 h at 37°C in a 5% CO2 humidified atmosphere. Collect the supernatant, centrifuged at 1,000 × g for 10 min to remove cellular debris, the conditioned medium (CM) was obtained by mixing supernatant and fresh complete medium 1:1 (v/v). For macrophage stimulation, RAW264.7 cells were treated with the prepared CM for specified durations under standard culture conditions. Macrophage polarization was detected by Flow Cytometry. RRM2 inhibitor recovery assay (1) in vitro: transwell migration assays were performed using 0.4 µm pore-size chambers (Corning, Glendale, AZ, USA). MC38 or CT26 cells transfected with shRNA plasmids were seeded in the upper chamber, while RAW264.7 macrophages were placed in the lower chamber. Tumor cells in the upper chamber were treated with RRM2 inhibitor at a concentration of 823 µM. Following 48 hours of co-culture, RAW264.7 macrophages were harvested for subsequent staining and flow cytometry analysis. (2) in vivo: Briefly, MC38 cells (4 × 10⁶ cells in 100 µL PBS) were intraperitoneally injected into 6-week-old male C57BL/6 mice. The experimental groups received RRM2 inhibitors at doses of 100 mg/kg or 50 mg/kg every other day, while the control group was administered an equivalent volume of DMSO. Approximately 15 days post-inoculation, tumour-bearing mice were euthanized to harvest peritoneal tumors. Statistical analysis Statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software, Inc., USA). Data are presented as mean ± standard deviation of biological replicate. Statistical significance was defined as *P < 0.05, **P < 0.01, and ***P < 0.001 for all measured parameters. Results CAP1 is downregulated in colorectal cancer and inhibits tumor growth in vivo To elucidate the role of CAP1 in colorectal cancer (CRC) progression, we first compared CAP1 transcript expression in human CRC tissues and adjacent normal tissues using the TCGA database (The Cancer Genome Atlas Program). This reveals significantly downregulated CAP1 expression in CRC tissues (Fig. S1 A, 1A). Moreover, higher CAP1 expression positively correlated with patient survival (Fig. 1 B). Consistent with these findings, WB and immunohistochemical staining of clinical samples demonstrated reduced CAP1 expression in tumour tissues compared to adjacent normal tissues (Fig. 1 C-D). To further investigate the functions of CAP1, we silenced Cap1 expression in mouse CRC cell lines (MC38 and CT26) using shRNA-encoding lentivirus. Successful knockdown was confirmed in both cell lines using two different shRNA designs (Fig. S1 B–D). Interestingly, CAP1 downregulation did not alter cell proliferation rates although it significantly enhanced cell migration in vitro (Fig. S1 E–G). To understand how CAP1 knockdown affects tumor growth in vivo, To validate our findings in vivo, we established subcutaneous mouse models by injecting shNC shCAP1-1, shCAP1-2 MC38 cells in immuno-competent C57BL/6 mice (n = 6 per group) and monitored tumor size over 10 days. In difference with the trend observed in vitro, CAP1 knockdown markedly promoted tumor growth in vivo (Fig. 1 E–G). Similarly, when intraperitoneally injected (n = 5 per group), CAP1-knockdown MC38 cells grew much more quickly than control cells (Fig. 1 H, I). The lack of correlation between in vitro and in vivo experiments led us to suspect of the implication of tumor microenvironment. To test this hypothesis, we assessed tumor progression in immuno-deficient nude mice by subcutaneously injecting shNC shCAP1-1, shCAP1-2 MC38 cells (n = 6 per group). Interestingly, CAP1 knockdown did not affect tumor growth (Fig. S1 H–J). In total, these results indicate that CAP1 is a tumor-suppressor, and its effect is mediated by the tumor immune microenvironment. CAP1 silencing in colorectal cancer cells promotes macrophage M2-type polarization The tumor immune microenvironment critically regulates colorectal cancer progression. Given our earlier finding that CAP1 knockdown accelerated CRC growth specifically in immuno-competent mice, we hypothesized that CAP1 modulates anti-tumor immunity. To test this hypothesis, we employed a peritoneal carcinomatosis model that permits facile evaluation of the tumor-immune crosstalk. Briefly, MC38 cells with CAP1 knockdown (shCAP1) and control (shNC) were intraperitoneal injected into C57BL/6 mice (n = 6 per group). Subsequently, we isolated epididymal adipose tissue macrophages—key immune sentinels in the peritoneal cavity and characterized their phenotypes using well-characterized surface biomarkers (Fig. 2 A, Fig. S2 A). The results of the pre-experiment revealed that the proportion of macrophages changed significantly on the third day after intraperitoneal injection of tumour cells(Fig. S2 B-D). Interestingly, CAP1 silencing significantly enhanced macrophage polarization towards the immuno-suppressive M2 phenotype but not the M1 phenotype (Fig. 2 B-C, S2E). In addition to the peritoneal model, we employed the subcutaneous tumor model to examine macrophage infiltration, the results of which show markedly enhanced infiltration of CD163⁺ M2 macrophages in CAP1-knockdown tumors (Fig. 2 D). To dissect the underlying mechanism, we used the transwell co-culture system to distinguishing contact-dependent from soluble-factor-mediated effects (Fig. 2 E). This co-culture model physically separated tumor cells (in the upper chamber) from RAW264.7 macrophages (in the lower chamber). In line with the in vivo observation, CAP1-deficient CRC cells robustly induced M2 macrophage polarization (Fig. 2 F-I). Crucially, this effect was recapitulated when macrophages were exposed to the conditioned media of CAP1-knockdown cells (Fig. 2 J-M), demonstrating that soluble factors alone are sufficient to drive M2 macrophage polarization. Collectively, these data establish that CAP1 loss in CRC cells affected M2 macrophage polarization through secreting soluble factors. CAP1 modulates M2 polarization through RRM2 upregulation and secretion. Having established that CAP1-deficient CRC cells secrete soluble factors driving M2 macrophage polarization, we next sought to identify the specific molecular mediator. Quantitative proteomic profiling of CAP1-knockdown MC38 cells revealed 817 differentially expressed proteins (216 upregulated, 601 downregulated) compared to control MC38 cells (Fig. S3 A-B), with further pathway enrichment implicating metabolic reprogramming and immune modulation (Fig. S3 C-D). Strikingly, ribonucleotide reductase regulatory subunit M2 (RRM2) emerged as one of the most significantly upregulated proteins (Fig. 3 A-B). Further analysis of the TCGA database uncovers elevated RRM2 transcripts in human CRC tissues (Fig. 3 C). Next, we measured RRM2 protein levels in the cells and conditioned media using western blot and ELISA approaches, respectively. It was shown that CAP1-knockdown led to elevation RRM2 protein in MC38 and CT26 cells (Fig. 3 D) as well as in the conditioned media (Fig. 3 E). To understand the effects of RRM2, we treated RAW264.7 macrophages with recombinant murine RRM2 (rRRM2) for 48h. It was found that rRRM2 induced M2 macrophage polarization (Fig. 3 F), recapitulating the effect observed with the secretome of CAP1-knockdown cells. To validate these observations in vivo, we detected the expression of relevant molecules using animal tumor tissue. We found that in the tumors with low expression of CAP1, the expression of RRM2 increased and the number of M2-type macrophages increased (Fig. 3 G). Generally, these results indicate that CAP1 knockdown enhances the secretion of RRM2 which drives M2 macrophage polarization. Alkbh5-mediated m6A modification is involved in the regulation of RRM2 expression by CAP1 Our previous findings established that CAP1 depletion in CRC cells promotes M2 macrophage polarization through upregulation of RRM2 protein. To elucidate the molecular mechanism underlying this regulation, we first confirmed that while RRM2 protein levels increased in CAP1-knockdown cells, quantitative PCR analysis does not show corresponding change in Rrm2 mRNA levels (Fig. 4 A), effectively excluding transcriptional regulation as the primary mechanism. This observation led us to systematically investigate alternative regulatory pathways. Initial examination of protein stability using cycloheximide chase assays reveals unchanged RRM2 degradation kinetics in CAP1-deficient cells (Fig. S4 ), directing our attention toward post-transcriptional regulatory mechanisms involving RNA modifications that influence translation efficiency. We hypothesized that CAP1 might regulate RRM2 through m6A-dependent translational control. This hypothesis was initially supported by our observation of significantly elevated global m6A levels in CAP1-knockdown cells (Fig. 4 B). Further analysis of the previous proteomics data revealed that in CAP1-knockdown cells, the expression level of ALKBH5, an eraser of m6A modification, decreased significantly (Fig. 4 C). The mRNA expression level of ALKBH5 was decreased in cells with cap1 low-expression (Fig. 4 D). Moreover, overexpression of ALKBH5 significantly reduced the protein expression of RRM2 (Fig. 4 E). Together, these results provide evidence that CAP1 regulates RRM2 protein levels through ALKBH5-dependent m6A modification pathway. The RRM2 inhibitor osalmid rescues CAP1-loss-driven tumor growth Having established RRM2 as the mechanistic mediator between CAP1 loss and M2 macrophage polarization (Fig. 3 ), we next investigated whether pharmacological inhibition of RRM2 could reverse this effect. Osalmid was previously reported to inhibit RRM2 [ 27 ] . Here, we first tested whether osalmid could inhibit M2 macrophage polarization and found that it significantly attenuated IL-4/IL-10-induced M2 polarization of RAW264.7 cells (Fig. 5 A-B). Next, we tested the efficacy of osalmid to reduce M2 macrophage polarization induced by CAP1-knockdown CRC cells. In the transwell co-culture system, osalmid added to the lower chamber reduced the percentage of CD11b-CD206 + cells to the basal level in MC38 (Fig. 5 C-D) and CT26 cells (Fig. S5A-B), demonstrating the therapeutic potential of inhibiting RRM2. Furthermore, we investigated the efficacy of osalmid in the peritoneal carcinomatosis model. The mice were randomly divided into 6 groups (n = 6 per group), including the control group, the CAP1 low-expression group, and the osalmid treatment group. Importantly, osalmid selectively inhibited growth of CAP1-knockdown tumors while showing no efficacy against control tumors (Fig. 5 E-F). A similar trend was observed in the analysis of tumor-associated macrophages phenotypes, which demonstrates a sharp decline in M2 macrophage polarization (Fig. 5 G-H). In conclusion, these results support that osalmid selectively inhibited the growth of CAP1-knockdown cells through reversing the immuno-suppressive phenotype of tumor-associated macrophages. Discussion Colorectal cancer has long posed a significant threat to human health and life, although immunotherapy combined with surgical resection and chemoradiotherapy has significantly improved patients survival, the outcomes for some patients remain suboptimal. Therefore, it is urgent to find new therapeutic targets. In this study, bioinformatics analysis revealed that CAP1 is markedly downregulated in tumors and exhibits a significant positive correlation with patient survival, indicating its potential functional involvement in the pathogenesis and progression of colorectal cancer. In recent years, many studies have found that CAP1 is highly associated with cancer, such as: breast cancer, liver cancer, lung cancer, etc. In breast cancer (BC), CAP1 is highly expressed and significantly correlates with BC grade, and promotes BC cell proliferation and migration through down-regulation of C-terminal Binding Protein 2 (CtBP2) and E-cadherin expression [ 28 , 29 ] . In addition, CAP1 is overexpressed in ovarian cancer (OC) and contributes to the cell cycle by affecting the cell cycle [ 30 ] . Although some studies suggest that CAP1 contributes to the development of various tumors, its role in colorectal cancer (CRC) remains less explored. Researchers have observed that CAP1 expression does not markedly rise in several CRC cell lines. Furthermore, CAP1 knockdown significantly boosts the migration of CRC cells without significantly affecting their proliferation [ 10 ] . Our results are consistent with these observations, as we too found that CAP1 knockdown markedly enhances CRC cell migration but has no significant impact on proliferation in CRC cell lines. However, in vivo experiments have shown that the absence of CAP1 can significantly promote tumor growth in immunocompetent mice, suggesting that CAP1's effect on CRC growth may be related to the tumor's immune microenvironment. Cancer is a group of diseases characterized by uncontrolled cell proliferation. In traditional tumor research, scientists focus on the effect of the intrinsic changes of cancer cells on tumor proliferation, migration, drug resistance, etc. However, there is increasing evidence showed that tumor cells reside in a complex microenvironment with diverse components, there are extensive interactions between cancer cells and the tumor microenvironment, and it is necessary to consider the tumor and its microenvironment as an integrated whole [ 13 ] . The interplay between tumor cells and immune cells in the microenvironment plays an important role in tumor growth. Studies have indicated that tumor cells upregulate the expression of PMVK, to stabilize GAD1 and recruit ACAT1, resulting in elevated levels of 4-Ac-GABA in the tumor microenvironment. The 4-Ac-GABA binds to GABAAR on CD8 + T cells, inhibiting the AKT1 signal pathway, which in turn suppresses the activation of CD8 + T cells and the anti-tumor response [ 31 ] . Our findings revealed that CAP1 markedly suppressed the polarization of macrophages towards the M2 phenotype, irrespective of whether the macrophages were isolated in vivo or co-cultured with tumour cells in vitro. N6-methyladenosine (m6A) RNA modification has emerged as a pivotal regulator in tumorigenesis and cancer progression. Accumulating evidence indicates that m6A plays a dual role in cancer, acting as either an oncogene or a tumor suppressor depending on the context [ 32 ] . For instance, elevated expression of certain m6A methyltransferases, such as ALKBH5 inhibited Gastric cancer invasion and migration by modulating the stability of PKMYT1 mRNAs [ 33 ] . Moreover, m6A is intricately involved in shaping the tumor immune microenvironment [ 34 ] . It can influence the infiltration and function of immune cells, thereby affecting tumor immune evasion. Our research results indicate that Alkbh5-mediated m6A modification of RRM2 mRNA significantly enhances the protein expression of RRM2 in CRC. RRM2 is associated with Ferroptosis in macrophages [ 35 ] , and promote polarization of macrophage to M2 macrophage [ 26 ] . Our research further substantiates that RRM2 can drive the polarization of macrophages towards the M2 phenotype. Moreover, osalmid, an inhibitor of RRM2, can significantly suppress the growth of colorectal cancer (CRC). These results highlight RRM2 inhibitors exhibit potent anticancer effects in CRC and RRM2 hold promise as potential therapeutic target for treating CRC. The present study uncovered a novel mechanism of action of CAP1 acting in RRM2 proteins, which is a novel finding, thus elucidating the mechanism by which CAP1 plays a role in colorectal cancer. Notably, the CAP1/RRM2 axis can be synergistically inhibited by conventional chemotherapeutic drug combinations that can inhibit CRC progression. In conclusion, we demonstrated that the CAP1/RRM2 axis in CRC cells regulates anti-tumor immunity through polarized macrophage M2 type. Our findings may provide new strategies for the clinical treatment of colorectal cancer. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Ethics approval Human study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Fourth People's Hospital of Chong qing (Chongqing Emergency Medical Center) (Approval Number: 2025 (54)). All animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of Chongqing University Central Hospital (Approval Number: 2412004) Consent to participate Informed consent was obtained from all individual participants included in the study Funding This work was supported by the Science and Technology Research Program of Chongqing Municipal Education Commission (Grant No. KJQN202400128), Chongqing Municipal Health Commission (Grant No 2024GDRC011) and Chongqing University (Grant No 2023CDJYGRH-YB10). Author Contributions Tianlin Feng: implementation of the research plan, participation in data analysis and paper writing. Ling Lin: experimental design, paper writing. Xiaoya Zhou and Li Li: cell experiments. Yao Chen and Wenyi Zheng: sample collection and processing. Qinrui Cai and Dongling Li: Data analysis and statistics. Jianshan Lin and Qianyao Wang: Literature review and background material organization. Wei Li and Xiaoyuan Zheng: experimental design and data analysis, providing professional opinions and technical support. Fan Yang: project initiation, research planning and design. All authors read and approved the final manuscript Data Availability Statement The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. The MS proteomics data have been deposited at the iProX database ( https://iprox.org ) with the program ID: PXD065883 References Hardcastle JD, Thomas WM, Chamberlain J et al (1989) Randomised, controlled trial of faecal occult blood screening for colorectal cancer. Results for first 107,349 subjects. Lancet 1(8648):1160–1164 Siegel RL, Giaquinto AN (2024) Jemal Cancer statistics, 2024. CA Cancer J Clin 74(1):12–49 Bray F, Laversanne M, Sung H et al (2024) Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 74(3):229–263 Ono S (2013) The role of cyclase-associated protein in regulating actin filament dynamics - more than a monomer-sequestration factor. J Cell Sci 126(Pt 15):3249–3258 Jansen S, Collins A, Golden L et al (2014) Structure and mechanism of mouse cyclase-associated protein (CAP1) in regulating actin dynamics. J Biol Chem 289(44):30732–30742 Yu G, Swiston J (1994) Young Comparison of human CAP and CAP2, homologs of the yeast adenylyl cyclase-associated proteins. J Cell Sci 107(Pt 6):1671–1678 Xue B (2013) Robinson Guardians of the actin monomer. Eur J Cell Biol 92(10–11):316–332 Shekhar S, Chung J, Kondev J et al (2019) Synergy between Cyclase-associated protein and Cofilin accelerates actin filament depolymerization by two orders of magnitude. Nat Commun 10(1):5319 Bergqvist M, Elebro K, Sandsveden M et al (2020) Effects of tumor-specific CAP1 expression and body constitution on clinical outcomes in patients with early breast cancer. Breast Cancer Res, 22(1) Ramsey A, Akana L, Miyajima E et al (2023) CAP1 (cyclase-associated protein 1) mediates the cyclic AMP signals that activate Rap1 in stimulating matrix adhesion of colon cancer cells. Cell Signal 104:110589 Lee S, Lee HC, Kwon YW et al (2014) Adenylyl cyclase-associated protein 1 is a receptor for human resistin and mediates inflammatory actions of human monocytes. Cell Metab 19(3):484–497 Zhu Y, Wan N, Shan X et al (2021) Celastrol targets adenylyl cyclase-associated protein 1 to reduce macrophages-mediated inflammation and ameliorates high fat diet-induced metabolic syndrome in mice. Acta Pharm Sin B 11(5):1200–1212 Xiao Y (2021) Yu Tumor microenvironment as a therapeutic target in cancer. Pharmacol Ther 221:107753 Di Martino JS, Mondal C (2019) Bravo-Cordero Textures of the tumour microenvironment. Essays Biochem 63(5):619–629 Osipov A, Murphy A (2019) Zheng From immune checkpoints to vaccines: The past, present and future of cancer immunotherapy. Adv Cancer Res 143:63–144 Cassetta L (2018) Pollard Targeting macrophages: therapeutic approaches in cancer. Nat Rev Drug Discov 17(12):887–904 DeNardo DG (2019) Ruffell Macrophages as regulators of tumour immunity and immunotherapy. Nat Rev Immunol 19(6):369–382 Watanabe S, Alexander M, Misharin AV et al (2019) The role of macrophages in the resolution of inflammation. J Clin Invest 129(7):2619–2628 Yeung OW, Lo CM, Ling CC et al (2015) Alternatively activated (M2) macrophages promote tumour growth and invasiveness in hepatocellular carcinoma. J Hepatol 62(3):607–616 Wang N, Liang H (2014) Zen Molecular mechanisms that influence the macrophage m1-m2 polarization balance. Front Immunol 5:614 Boutilier AJ (2021) and S.F. Elsawa Macrophage Polarization States in the Tumor Microenvironment. Int J Mol Sci, 22(13) Zuo Z, Zhou Z, Chang Y et al (2024) Ribonucleotide reductase M2 (RRM2): Regulation, function and targeting strategy in human cancer. Genes Dis 11(1):218–233 D'Angiolella V, Donato V, Forrester FM et al (2012) Cyclin F-mediated degradation of ribonucleotide reductase M2 controls genome integrity and DNA repair. Cell 149(5):1023–1034 Xiong W, Zhang B, Yu H et al (2021) RRM2 Regulates Sensitivity to Sunitinib and PD-1 Blockade in Renal Cancer by Stabilizing ANXA1 and Activating the AKT Pathway. Adv Sci (Weinh) 8(18):e2100881 Tang B, Xu W, Wang Y et al (2021) Identification of critical ferroptosis regulators in lung adenocarcinoma that RRM2 facilitates tumor immune infiltration by inhibiting ferroptotic death. Clin Immunol, 232 Tang B, Xu W, Wang Y et al (2021) Identification of critical ferroptosis regulators in lung adenocarcinoma that RRM2 facilitates tumor immune infiltration by inhibiting ferroptotic death. Clin Immunol 232:108872 Tang Q, Wu L, Xu M et al (2020) Osalmid, a Novel Identified RRM2 Inhibitor, Enhances Radiosensitivity of Esophageal Cancer. Int J Radiat Oncol Biol Phys 108(5):1368–1379 Liu X, Yao N, Qian J et al (2014) High expression and prognostic role of CAP1 and CtBP2 in breast carcinoma: associated with E-cadherin and cell proliferation. Med Oncol 31(3):878 Yu XF, Ni QC, Chen JP et al (2014) Knocking down the expression of adenylate cyclase-associated protein 1 inhibits the proliferation and migration of breast cancer cells. Exp Mol Pathol 96(2):188–194 Hua M, Yan S, Deng Y et al (2015) CAP1 is overexpressed in human epithelial ovarian cancer and promotes cell proliferation. Int J Mol Med 35(4):941–949 Zhou X, Chen Z, Yu Y et al (2024) Increases in 4-Acetaminobutyric Acid Generated by Phosphomevalonate Kinase Suppress CD8(+) T Cell Activation and Allow Tumor Immune Escape. Adv Sci (Weinh) 11(43):e2403629 Wang T, Kong S, Tao M et al (2020) The potential role of RNA N6-methyladenosine in Cancer progression. Mol Cancer 19(1):88 Hu Y, Gong C, Li Z et al (2022) Demethylase ALKBH5 suppresses invasion of gastric cancer via PKMYT1 m6A modification. Mol Cancer, 21(1) Zhao L, Li Q, Zhou T et al (2024) Role of N6-methyladenosine in tumor neovascularization. Cell Death Dis 15(8):563 Tang B, Zhu J, Wang Y et al (2023) Targeted xCT-mediated Ferroptosis and Protumoral Polarization of Macrophages Is Effective against HCC and Enhances the Efficacy of the Anti-PD-1/L1 Response. Adv Sci (Weinh) 10(2):e2203973 Statements & Declarations Supplementary Files EthicalapprovalforthehumanstudyNo54.pdf WBoriginaldatafile.pdf ethicalapprovalforanimalstudy.pdf supplementmaterials.pdf Cite Share Download PDF Status: Published Journal Publication published 02 Jan, 2026 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted Editorial decision: Major Revision 17 Oct, 2025 Reviewers agreed at journal 01 Oct, 2025 Reviewers invited by journal 17 Sep, 2025 Editor assigned by journal 16 Aug, 2025 First submitted to journal 14 Aug, 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7355735","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":516536459,"identity":"ca36a2fc-2b61-4481-9fae-fa0353a0ed22","order_by":0,"name":"Tianlin Feng","email":"","orcid":"","institution":"Chongqing University Central Hospital: Chongqing Emergency Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Tianlin","middleName":"","lastName":"Feng","suffix":""},{"id":516536460,"identity":"73c73c3d-e34a-458a-90c8-bb97f8368735","order_by":1,"name":"Ling Lin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAw0lEQVRIiWNgGAWjYBACPmYGxgMJDAxyEC4bEVrYmBkYQFqMSdACxAeAOLGBeC3svAcOPNxRm97fnmPA8KHsMAP/7AZCDuNLOJB45njujDNvDBhnnDvMIHHnACEtPAYHEtuO5W6QyDFg5m07zGAgkUCclnQDkJa/JGipSQBrYSRBywHDGWeeFRzsOZfOI3GDgBZ+/jOGD3+21cnztydvfPCjzFqOfwYBLVBwGIgTwBHEQ5R6IKgDaxkFo2AUjIJRgBUAAN4SQD25VmBTAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0009-0007-8973-4412","institution":"Chongqing University Central Hospital: Chongqing Emergency Medical Center","correspondingAuthor":true,"prefix":"","firstName":"Ling","middleName":"","lastName":"Lin","suffix":""},{"id":516536461,"identity":"8172a974-8843-482a-8d91-480a7b20b257","order_by":2,"name":"Xiaoya Zhou","email":"","orcid":"","institution":"Chongqing University Central Hospital: Chongqing Emergency Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Xiaoya","middleName":"","lastName":"Zhou","suffix":""},{"id":516536462,"identity":"ce6ca42e-c8aa-4873-9176-9b16c510704c","order_by":3,"name":"Li Li","email":"","orcid":"","institution":"Chongqing University Central Hospital: Chongqing Emergency Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Li","suffix":""},{"id":516536463,"identity":"88f616a0-1d0f-4858-aa6c-a835f904356e","order_by":4,"name":"Yao Chen","email":"","orcid":"","institution":"Chongqing University Central Hospital: Chongqing Emergency Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Chen","suffix":""},{"id":516536464,"identity":"b77e8c6d-af35-48da-bdad-245d99b33027","order_by":5,"name":"Wenyi Zheng","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenyi","middleName":"","lastName":"Zheng","suffix":""},{"id":516536465,"identity":"0c9bd8aa-aa00-48ff-b0fc-2a9963354efc","order_by":6,"name":"Qinrui Cai","email":"","orcid":"","institution":"Chongqing University Central Hospital: Chongqing Emergency Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Qinrui","middleName":"","lastName":"Cai","suffix":""},{"id":516536466,"identity":"e7199716-e9c2-46a1-a830-92173529f9b3","order_by":7,"name":"Dongling Li","email":"","orcid":"","institution":"Chongqing University Central Hospital: Chongqing Emergency Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Dongling","middleName":"","lastName":"Li","suffix":""},{"id":516536467,"identity":"8bc4e992-a84b-49ff-a4ef-dd2acc7bdded","order_by":8,"name":"Jianshan Lin","email":"","orcid":"","institution":"Chongqing University Central Hospital: Chongqing Emergency Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Jianshan","middleName":"","lastName":"Lin","suffix":""},{"id":516536468,"identity":"c1bafb78-bada-4b29-be89-b831a6fd36ff","order_by":9,"name":"Qianyao Wang","email":"","orcid":"","institution":"Chongqing University Central Hospital: Chongqing Emergency Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Qianyao","middleName":"","lastName":"Wang","suffix":""},{"id":516536469,"identity":"c4cdb390-731a-4a10-8b6b-b4ed0eb71645","order_by":10,"name":"Wei Li","email":"","orcid":"","institution":"Chongqing University Central Hospital: Chongqing Emergency Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Li","suffix":""},{"id":516536470,"identity":"825a778d-27c1-4dc1-a36e-9f2a06a29baa","order_by":11,"name":"Xiaoyuan Zheng","email":"","orcid":"","institution":"Chongqing University Central Hospital: Chongqing Emergency Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyuan","middleName":"","lastName":"Zheng","suffix":""},{"id":516536471,"identity":"86131994-3847-4e08-b22e-411e98ac12bb","order_by":12,"name":"Fan Yang","email":"","orcid":"https://orcid.org/0009-0001-5135-1279","institution":"Chongqing University Central Hospital: Chongqing Emergency Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Fan","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-08-12 12:30:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7355735/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7355735/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00018-025-06031-x","type":"published","date":"2026-01-02T15:57:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":92260264,"identity":"a33404ce-fac9-443b-bd6f-7402a527aa36","added_by":"auto","created_at":"2025-09-26 12:26:50","extension":"tif","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6344276,"visible":true,"origin":"","legend":"","description":"","filename":"figure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/809f4e0a16cd59d5a0f8d08e.tif"},{"id":92259073,"identity":"503b85b6-38b6-43b1-8ff2-41598013a3cf","added_by":"auto","created_at":"2025-09-26 12:10:49","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4762892,"visible":true,"origin":"","legend":"","description":"","filename":"figure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/dd944055d6772fb08af1f2be.tif"},{"id":92259394,"identity":"a75069fd-6493-45c1-9959-810b4064acd2","added_by":"auto","created_at":"2025-09-26 12:18:50","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7187816,"visible":true,"origin":"","legend":"","description":"","filename":"figure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/c7209a693f501861119d57e4.tif"},{"id":92259072,"identity":"6b84c6c5-5e4c-4cca-b18e-cc61c46ea7cf","added_by":"auto","created_at":"2025-09-26 12:10:49","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1303540,"visible":true,"origin":"","legend":"","description":"","filename":"figure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/9454d8c0d7481a2644077737.tif"},{"id":92259077,"identity":"eb79cc0d-12d6-40eb-8a02-78026edda640","added_by":"auto","created_at":"2025-09-26 12:10:50","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":15910220,"visible":true,"origin":"","legend":"","description":"","filename":"figure5.tif","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/e11c838882f2b0523a8ddfeb.tif"},{"id":92259089,"identity":"5c648f15-6ad0-4120-8e34-cb2f7bce8deb","added_by":"auto","created_at":"2025-09-26 12:10:50","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2218032,"visible":true,"origin":"","legend":"","description":"","filename":"figure6.tif","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/bce5d178b8f5f72bfa2705ce.tif"},{"id":92259078,"identity":"4ea0a9c3-0ef9-47ef-b5cb-9e24c76b8a71","added_by":"auto","created_at":"2025-09-26 12:10:50","extension":"xml","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":17554,"visible":true,"origin":"","legend":"","description":"","filename":"lifeLIFED2502152.xml","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/40c6a4a5446c1b2291280601.xml"},{"id":92259080,"identity":"26b0876c-39b4-4e8f-bc28-d4b0c29733e7","added_by":"auto","created_at":"2025-09-26 12:10:50","extension":"xml","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1352,"visible":true,"origin":"","legend":"","description":"","filename":"LIFED250215254292.go.xml","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/152ae8d51a3999a5ff44b231.xml"},{"id":92259076,"identity":"b6b56fc2-5d84-430e-bb25-ba6f0ec3289d","added_by":"auto","created_at":"2025-09-26 12:10:50","extension":"xml","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":931,"visible":true,"origin":"","legend":"","description":"","filename":"LIFED2502152Import.xml","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/ed6735bab5a3e029487c5006.xml"},{"id":92259083,"identity":"fbf963aa-84c4-4dbb-92e2-22de3a8ec0f2","added_by":"auto","created_at":"2025-09-26 12:10:50","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":99082,"visible":true,"origin":"","legend":"","description":"","filename":"LIFED25021520enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/ebe8bfe1768e2e241b89097a.xml"},{"id":92259400,"identity":"60e03e84-6b70-4bc9-923e-c9dfdc048dba","added_by":"auto","created_at":"2025-09-26 12:18:50","extension":"tif","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6344276,"visible":true,"origin":"","legend":"","description":"","filename":"figure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/2044d14beac14bf6f6b47f24.tif"},{"id":92259100,"identity":"e69acd73-5719-438e-a65f-7cc69a4dace4","added_by":"auto","created_at":"2025-09-26 12:10:50","extension":"tif","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4762892,"visible":true,"origin":"","legend":"","description":"","filename":"figure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/bd26510b50b517a7382f6c91.tif"},{"id":92259099,"identity":"ab8b49ac-2e03-4026-9049-779758982d76","added_by":"auto","created_at":"2025-09-26 12:10:50","extension":"tif","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7187816,"visible":true,"origin":"","legend":"","description":"","filename":"figure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/8ae3787a4023b91b9df61ad8.tif"},{"id":92259091,"identity":"2f509e81-192b-47a4-a444-4bd927ab2762","added_by":"auto","created_at":"2025-09-26 12:10:50","extension":"tif","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1303540,"visible":true,"origin":"","legend":"","description":"","filename":"figure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/f2770e36bc236447f0ce9519.tif"},{"id":92259101,"identity":"bbbb346b-216a-43b7-b67c-8967a244ea39","added_by":"auto","created_at":"2025-09-26 12:10:50","extension":"tif","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":15910220,"visible":true,"origin":"","legend":"","description":"","filename":"figure5.tif","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/a8e9f8b429e5961f1d1b3e03.tif"},{"id":92259085,"identity":"47fd4481-9ffd-4491-9da5-1fd89e2a1433","added_by":"auto","created_at":"2025-09-26 12:10:50","extension":"tif","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2218032,"visible":true,"origin":"","legend":"","description":"","filename":"figure6.tif","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/04bb24b530365c873bdfac5e.tif"},{"id":92259095,"identity":"ee88f2b1-2c03-4ebd-9a4a-09c0bd383135","added_by":"auto","created_at":"2025-09-26 12:10:50","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":395171,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/49bf974ee14891c78b793b8f.png"},{"id":92259088,"identity":"b79898df-e9db-47d1-8761-2b25bbfa03f5","added_by":"auto","created_at":"2025-09-26 12:10:50","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":365276,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/bc9e66ec227723743ee01357.png"},{"id":92259397,"identity":"0b286edb-0378-4463-8e62-cc0c1af2cf34","added_by":"auto","created_at":"2025-09-26 12:18:50","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":499909,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/7209a67b8486d09228d94f7b.png"},{"id":92259401,"identity":"8ac7f45a-dc91-4963-b004-e6b4dc15ef8d","added_by":"auto","created_at":"2025-09-26 12:18:50","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":60742,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/455b79feed05d55d7d4cda58.png"},{"id":92259096,"identity":"fc35c808-0326-4862-ba30-efd9a4b01ebb","added_by":"auto","created_at":"2025-09-26 12:10:50","extension":"png","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":732896,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/b9f8b78994a7f6c96ea2e9c3.png"},{"id":92259084,"identity":"0b4a6cf4-cb4a-4091-967b-a3c363e73d71","added_by":"auto","created_at":"2025-09-26 12:10:50","extension":"png","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1140229,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/1073408808c98dbd957accef.png"},{"id":92259398,"identity":"bf6b7817-7ab9-4acb-bb9f-c416223206dc","added_by":"auto","created_at":"2025-09-26 12:18:50","extension":"xml","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":97479,"visible":true,"origin":"","legend":"","description":"","filename":"LIFED25021520structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/e99579ad9aa97425cf04b1e7.xml"},{"id":92259092,"identity":"a065850a-84f5-4da6-abd4-3d5466e87566","added_by":"auto","created_at":"2025-09-26 12:10:50","extension":"html","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":107012,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/5005055494366de3ea44b44d.html"},{"id":92259068,"identity":"d956d353-29b1-473a-948e-71e17d6d3c53","added_by":"auto","created_at":"2025-09-26 12:10:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11584950,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCAP1 is downregulated in colorectal cancer and inhibits tumor growth in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) CAP1 transcript expression in CRC versus normal tissues (TCGA analysis). (B) Kaplan-Meier survival analysis of CRC patients stratified by CAP1 expression (TCGA data). (C) WB validation of CAP1 downregulation in clinical CRC tissues versus matched adjacent normal tissues. (D) Representative immunohistochemical staining of CAP1 in clinical CRC and adjacent normal tissues. (E-G) Subcutaneous xenograft model in C57BL/6 mice: (E) Representative tumor images at endpoint (day 10). (F) Tumor growth kinetics measured every 2-3 days. (G) Final tumor weights. (H-I) Peritoneal xenograft model in C57BL/6 mice. (H)Peritoneal tumors at endpoint (day 15). (I) Tumor weights. Data represent the mean ± standard deviation of biological replicates. ns. non-significant; *P \u0026lt; 0.05; **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/5b1096cec9e960ee67549831.png"},{"id":92259393,"identity":"67eddb8e-ff17-4f50-b3e3-92ea44c53148","added_by":"auto","created_at":"2025-09-26 12:18:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8175565,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCAP1 silencing in colorectal cancer cells promotes macrophage M2-type polarization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Scheme of isolation and polarization analysis of peritoneal macrophages. (B-C) Flow cytometry of M1/M2 macrophages from epididymal fat in peritoneal models. (D) CD163 immunohistochemistry of subcutaneous tumors from Fig. 1E. (E)Transwell co-culture system where tumor cells and RAW264.7 cells were seeded at the upper and lower chamber, respectively. (F-G) M2 polarization of RAW264.7 after co-culture with CAP1-knockdown MC38 (F) or CT26 (G) cells. (H-I) Quantification of (F) and (G). (J-K) M2 polarization of RAW264.7 treated with conditioned medium from CAP1-knockdown MC38 (J) or CT26 (K) cells. (L-M) Quantification of (J) and (K). Macrophage gating: In vivo: M1 (CD45⁺F4/80⁺CD11b⁺CD206⁻), M2 (CD45⁺F4/80⁺CD11b⁺CD206⁺); RAW264.7: M1 (CD45⁺CD11b⁺CD206⁻), M2 (CD45⁺CD11b⁺CD206⁺). Data represent mean ± standard deviation of biological replicates. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/550a44b04376a92ca61c1ee3.png"},{"id":92259396,"identity":"58c75ad4-db72-4def-8195-6141a7647e46","added_by":"auto","created_at":"2025-09-26 12:18:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":12355679,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCAP1 modulates M2 polarization through RRM2 upregulation and secretion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A): scatter plot of proteomic changes in CAP1-knockdown vs control MC38 cells. (B): Mass spectrometry quantification of RRM2 upregulation. (C): RRM2 transcript expression in human CRC (TCGA). (D): RRM2 protein elevation in CAP1-knockdown cell lines. (E): Quantification of RRM2 protein in conditioned media using ELISA. (F): Recombinant RRM2-induced M2 polarization in RAW264.7 macrophages. (G): Representative image of the expression of CAP1, RRM2 and CD163 proteins in mouse tumors detected by immunohistochemistry Data represent mean ± standard deviation of biological replicates. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/645db68ea599758d914a87aa.png"},{"id":92259069,"identity":"11134a64-0f55-4f48-a986-81552ca9b208","added_by":"auto","created_at":"2025-09-26 12:10:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1185764,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eALKBH5-mediated m6A modification is involved in the regulation of RRM2 expression by CAP1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A): qPCR detection of RRM2 expression levels in CAP1 knockdown cell lines. (B): protein m6A modification level in CAP1 knockdown cell lines in dot blot experiments. (C): Mass spectrometry quantification of RRM2 upregulation and ALKBH5 downregulation. (D): qPCR validation of Alkbh5 expression in cells with CAP1 knockdown. (E): Western blot analysis was performed to detect the protein expression of RRM2 in MC38 cells with overexpression of ALKBH5. ns, no significant; *, P＜0. 05; **, P＜0. 01; ***, P＜0. 001.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/470875a58b07f12b6f5e2faf.png"},{"id":92259086,"identity":"1171d83e-7750-470d-bc42-b3aa09a40ff6","added_by":"auto","created_at":"2025-09-26 12:10:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":31620638,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe RRM2 inhibitor osalmid rescues CAP1-loss-driven tumor growth\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B): Osalmid blocks cytokine-induced M2 polarization in RAW264.7 macrophages. (C-D): Osalmid rescues M2 polarization when co-culturing macrophages and CAP1-knockdown-MC38. (E-F): Osalmid selectively inhibits CAP1-deficient tumor growth in peritoneal models. (G-H): Osalmid reverses CAP1-loss-driven M2 polarization in tumor-associated macrophages. Data represent the mean ± standard deviation of biological replicates. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001 vs respective controls; #P \u0026lt; 0.05 for indicated comparisons.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/0284f7b51f921c86b5fe0a5a.png"},{"id":92259071,"identity":"8e0f2b13-1071-452a-aba2-b77aa4bfd60d","added_by":"auto","created_at":"2025-09-26 12:10:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3661976,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanism diagram of CAP1 regulation of colorectal cancer growth\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/5b05b05c939dacc1589e2ead.png"},{"id":99545203,"identity":"f20c2fe2-3588-446c-9a72-8592a4b486d1","added_by":"auto","created_at":"2026-01-05 16:02:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":63721877,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/33be19ce-02e4-4c06-8ce9-acee31a493cd.pdf"},{"id":92259399,"identity":"0c5cb10b-a67f-43d1-96ba-f330145ff3f0","added_by":"auto","created_at":"2025-09-26 12:18:50","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":1030769,"visible":true,"origin":"","legend":"","description":"","filename":"EthicalapprovalforthehumanstudyNo54.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/bf1485913b122e9204649255.pdf"},{"id":92259081,"identity":"371564b2-47d0-4042-a766-9575723cdbe6","added_by":"auto","created_at":"2025-09-26 12:10:50","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":143728,"visible":true,"origin":"","legend":"","description":"","filename":"WBoriginaldatafile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/ca5c059c7dd620fe7583ab2a.pdf"},{"id":92259093,"identity":"62bee2a5-5986-4375-b1b6-753775884608","added_by":"auto","created_at":"2025-09-26 12:10:50","extension":"pdf","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":464432,"visible":true,"origin":"","legend":"","description":"","filename":"ethicalapprovalforanimalstudy.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/3cb04b469f2494ab99b1d2cd.pdf"},{"id":92259087,"identity":"2e1cd365-1cee-419c-95cb-2555ad3a49b1","added_by":"auto","created_at":"2025-09-26 12:10:50","extension":"pdf","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":1176101,"visible":true,"origin":"","legend":"","description":"","filename":"supplementmaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7355735/v1/5331165c0bb772c8f98f00d7.pdf"}],"financialInterests":"","formattedTitle":"CAP1-Mediated m6A Modification of RRM2 Suppresses Tumor-Associated M2 Macrophage Polarization and Colorectal Cancer Growth","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer (CRC) is one of the most common malignant tumors, with the second highest number of new diagnoses in women and the third highest in men, and causing approximately 90,000 deaths annually worldwide\u003csup\u003e[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Advancements in medical science have led to significant improvements in the clinical management of colorectal cancer (CRC). Traditional treatments, including surgical resection, chemotherapy and radiotherapy, combined with emerging therapies such as targeted therapy and immunotherapy have collectively enhanced patient outcomes. Nevertheless, these treatments still face many challenges including high recurrence rates, severe adverse reactions, inadequate drug specificity, and the development of resistance. Consequently, reveal the molecular mechanisms of CRC pathogenesis and identify novel therapeutic targets are essential to further improve clinical efficacy and patient prognosis.\u003c/p\u003e\u003cp\u003eCyclase-associated protein (CAP) was first identified in yeast as an adenylyl cyclase-associated binding protein with two mammalian homologs, CAP1 and CAP2. CAP2 is a highly structured multifunctional protein with six structural domains\u003csup\u003e[\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Canonically, it regulates the actin cytoskeleton and Ras regulation of adenylyl cyclase\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. However, recent studies shown that CAP1 plays an important role in the development of tumors, in breast cancer patients, the expression level of CAP1 was significantly positively correlated with the survival time of patients\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. CAP1 plays critical role in linking the major second messenger cAMP to the activation of adherent Rap1 in colon cancer cells, which may also regulate the proliferation of other cell types\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. as a receptor of human resistin, CAP1 is involved in immune cell-mediated inflammation by upregulating the concentration of cyclic AMP (cAMP) concentration, protein kinase A (PKA) activity, and NF-κB-related transcription of inflammatory cytokines\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. However, the mechanism of CAP1 in the immune microenvironment of CRC has not been thoroughly studied.\u003c/p\u003e\u003cp\u003eThe interactions between cancer cells and tumor microenvironment (TME) are dynamic and reciprocal. Tumor microenvironment includes all non-cancerous host cells, including fibroblasts, endothelial cells, neurons, adipocytes, adaptive and innate immune cells, as well as its non-cellular components, including the extracellular matrix, and soluble products such as chemokines, cytokines, growth factors, and extracellular vesicles\u003csup\u003e[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Tumor-associated macrophages (TAM) are abundantly present in tumor microenvironment of most cancer types and the interaction between cancer cells and TAM shapes the tumor immune landscape are usually associated with clinical prognosis in cancer patients\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Macrophages in the tumor immune microenvironment can be divided into two subtypes: M1-type macrophages mainly play a role in tumor suppression, promotion of inflammation, and immunoreactivity, whereas M2-type macrophages play a role in tissue repair, immune escape, and promotion of tumorigenesis. The proportion of macrophages in tumor microcircuits is highly correlated with tumorigenesis, severity, and prognosis\u003csup\u003e[\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eRibonucleotide reductase M2 (RRM2) catalyzes the formation of deoxyribonucleotides from ribonucleotides\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. studies have shown that RRM2 plays an important role in tumor cell growth\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, drug resistance and immunotherapy\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Xiong et al found that RRM2 stabilizes ANXA1 and activates the AKT pathway independent of its ribonucleotide reductase activity, promoting sunitinib resistance in RCC. Moreover, RRM2 affects the anti-tumor effect of PD-1 antibody by regulating the expression of PD-L1 in tumor cells. At the same time, the expression of RRM2 is positively correlated with the infiltration of M2 macrophages in tumor tissues, and further promotes the growth of tumors\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. inhibition of RRM2 promotes macrophage M1 polarization and inhibits M2 polarization in lung adenocarcinoma\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. More importantly, RRM2 inhibitor Osalmid significantly enhances Radiosensitivity of Esophageal Cancer and suppressed tumor growth\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, we have demonstrated that CAP1 is lowly expressed in CRC and inhibits tumor growth and regulating the CRC growth through modulating the tumor immune microenvironment, Mechanistically, CAP1 regulates the expression and secretion of RRM2 protein through ALKBH5-mediated m6A modification, thereby affecting the polarization of M2 macrophages and inhibiting the growth of tumors, Moreover the combination of RRM2 inhibitor osalmid and low expression of CAP1 significantly inhibits the growth of CRC. The role of the CAP1/RRM2 axis has potential clinical applications and provides a new strategy for the comprehensive treatment of CRC(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell culture\u003c/h2\u003e\u003cp\u003eMC38 and CT26 were purchased from the Cell Resource Center, Institute of Basic Medical Sciences, CAMS/PUMC. All cell lines were authenticated using short tandem repeat (STR) profiling and routinely tested for mycoplasma contamination. Primary macrophages and cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and maintained at 37\u0026deg;C in a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePatient Tissue Specimens\u003c/h3\u003e\n\u003cp\u003eWith the consent of the patients, a total of 11 cases of resected specimens from colorectal cancer patients were collected for this study from February 2025 to May 2025. colorectal cancer specimens were compared with the paired adjacent cancer tissues from the same patient. and the study was approved by Ethics Committee of Fourth People's Hospital of Chong qing (Chongqing Emergency Medical Center) (Approval Number: 2025 (54)).\u003c/p\u003e\n\u003ch3\u003eMice\u003c/h3\u003e\n\u003cp\u003e All animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of Chongqing University Central Hospital (Approval Number: 2412004), Six-week-old male C57BL/6 mice and BALB/c Nude mice were purchased from Jiangsu Huachuang sino Pharma Tech(Jiangsu china) and housed in pathogen-free ventilated cages under controlled conditions (12 h light/dark cycle, 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 50\u0026thinsp;\u0026plusmn;\u0026thinsp;5% humidity) with ad libitum access to autoclaved food and sterile water. Only healthy mice, free from infectious diseases and parasites, were selected, and mice with health issues, abnormal weight changes were excluded. The sample size was chosen based on the literature and our previous experience. mice randomly assigned to different groups. For the peritoneal metastasis model, mice were intraperitoneally injected with 4 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e MC38 or CT26 cells suspended in 100 \u0026micro;L PBS. After 14 days, mice were euthanized and peritoneal tumor nodules were excised and weighed. For the subcutaneous tumor model, mice received a subcutaneous injection of 4 \u0026times; 10^6 MC38 cells in 100 \u0026micro;L PBS, and tumor-bearing mice were sacrificed on day 14 or 18 for tumor collection and weight measurement. All the mice were euthanized by cervical dislocation after isoflurane anesthesia, and the tumors were collected.\u003c/p\u003e\n\u003ch3\u003eEstablishment of CAP1-silenced MC-38 or CT-26 cell lines\u003c/h3\u003e\n\u003cp\u003eLentiviral vectors for CAP1 knockdown were generated by Genechem. (Shanghai, China). The following shRNA sequences were used: shCAP1-1 (5'-GGCTTACATCAAGGAGTTT-3'), shCAP1-2 (5'-TCTACCTTTCTGCTCTCTTAA-3'), and negative control shRNA (shNC, 5'-TTCTCCGAACGTGTCACGT-3'). The lentivirus were employed to infect MC38 and CT26 cells at a multiplicity of infection (MOI) of 10. After 48 hours incubation, the culture medium was supplemented with 5 \u0026micro;g/mL puromycin to selectively screen for successfully transduced cells. The knockdown efficiency of the target gene CAP1 was subsequently assessed using RT-qPCR and western blot.\u003c/p\u003e\n\u003ch3\u003eELISA\u003c/h3\u003e\n\u003cp\u003eThe medium supernatant levels of RRM2 were quantified using commercial ELISA kits (mlbio, Shanghai, China) following the manufacturer's protocol. Briefly, microplates were equilibrated at room temperature for 1 min before use. Standard solutions (50 \u0026micro;L/well) were added to designated wells in duplicate, while 50 \u0026micro;L of each sample was loaded into sample wells. Blank wells received nothing. After adding 100 \u0026micro;L of horseradish peroxidase (HRP)-conjugated detection antibody to all wells except blanks, plates were sealed and incubated at 37\u0026deg;C for 60 min. Following incubation, wells were emptied and washed five times with 350 \u0026micro;L washing buffer (1 min per wash). Then, 50 \u0026micro;L of chromogenic substrates A and B were added to each well, followed by incubation for 15 min at 37\u0026deg;C. The reaction was terminated by adding 50 \u0026micro;L stop solution, and absorbance was measured at 450 nm within 15 min using a microplate reader. A standard curve was generated by plotting the mean optical density values against corresponding standard concentrations, and sample concentrations were calculated using the linear regression equation derived from the standard curve.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eWestern blotting\u003c/h2\u003e\u003cp\u003eCells were washed three times with ice-cold PBS, followed by lysis in RIPA buffer supplemented with protease and phosphatase inhibitors (MCE, Shanghai, China). Protein concentrations were determined using the BCA Protein Assay Kit, with absorbance measured at 562 nm using a microplate reader (Epizyme biotech, Shanghai, China). Equal amounts of protein (30 \u0026micro;g) were separated by 10% SDS-PAGE and transferred onto PVDF membranes at appropriate voltages based on target protein molecular weights. Membranes were blocked with 5% skim milk in TBST at room temperature, followed by incubation with primary antibodies (Supplementary Table\u0026nbsp;1) at 4\u0026deg;C overnight. After three 5-min TBST washes, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Following another three TBST washes, protein bands were visualized using enhanced chemiluminescence substrate (Ncmbio, Suzhou, china) and imaged using a chemiluminescence detection system (Vilber, Paris, France).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eRNA m6A Dotblot Assays\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted using the Pure RNA Isolation Kit (Ncmbio, Suzhou, china) and diluted into a gradient concentration of 400 ng/\u0026micro;L, 200 ng/\u0026micro;L, 100 ng/\u0026micro;L and 50 ng/\u0026micro;L. Samples (400 ng, 200 ng, 100 ng, and 50 ng) that degenerated under 95\u0026deg; for 3 min were deposited on an Nitrocellulose membrane (ABclonal, Wuhan, China). Then, the membrane was crosslinked by ultraviolet rays for 5 min and washed with PBST. Subsequently, the membrane was blocked in 5% non-fat milk at room temperature for 1 hour and incubated with primary m6A antibody (ABclonal, Wuhan, China) overnight at 4 C. Dot blots were visualized after incubation with HRP-conjugated secondary antibody. After photography the membrane was stained with 0.02% methylene blue (Solarbio Beijing China) and washed with ddH\u003csub\u003e2\u003c/sub\u003eO until the background turned white, and then photographed.\u003c/p\u003e\n\u003ch3\u003eQuantitative real-time PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted using the Pure RNA Isolation Kit (Ncmbio, Suzhou, china) following the manufacturer's instructions. RNA concentration and purity were determined by NanoDrop One (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was synthesized from 1 ug total RNA using the ABScript III RT Master Mix for Qpcr with gDNA Remover according to the manufacturer's protocol (ABclonal, Wuhan, China). Quantitative real-time PCR (qPCR) was performed using 2X Universal SYBR Green Fast qPCR Mix (ABclonal, Wuhan, China) with each reaction containing 1 \u0026micro;L of cDNA template in a final volume of 20 \u0026micro;L. Quantitative PCR was performed using a PCR system(Bio-Rad, Hercules, CA, USA) and relative expression was calculated by the 2\u003csup\u003e(\u0026minus;∆∆Ct)\u003c/sup\u003e method. The primers were listed in Supplementary Table\u0026nbsp;2.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemistry\u003c/h2\u003e\u003cp\u003e Tumor tissue specimens were collected in accordance with protocols approved by the Scientific Research Committee of The Fourth People's Hospital of Chongqing, following ethical guidelines for human subject research. All (CRC) samples were obtained from treatment-naive patients undergoing surgical resection, with proper anonymization procedures. Tissues were fixed in 10% neutral buffered formalin and embedded in paraffin using standard protocols. Immunohistochemistry was performed according to manufacturer's instructions (Zsbio, Beijing, China). Briefly, 4-\u0026micro;m thick sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Antigen retrieval was performed by microwave heating in citrate buffer (pH 6.0) for 10 min. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide at room temperature for 10 min. Non-specific binding was blocked with 10% normal goat serum for 30 min at 37\u0026deg;C. Sections were then incubated with primary antibody against CAP1 overnight at 4\u0026deg;C. After washing with PBS, sections were incubated with HRP-conjugated secondary antibody for 20 min at 37\u0026deg;C, Signals were visualized with diaminobenzidine followed by hematoxylin counterstaining, dehydrated in ethanol, cleared in xylene, and mounted.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003eIsolation of fat macrophages from epididymal adipose tissue\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eMice were sacrificed via cervical vertebrae dislocation after isoflurane anesthesia. The epididymal adipose tissue was collected, cut and added into DMEM medium supplemented with 1 mg/ml collagenase IV (Sangon Biotech, China), 3% fetal bovine serum and 2% penicillin streptomycin, and digested at 37\u0026deg; for 1h. The digestive solution was filtered through 200 mesh sieve and centrifuged at 500g for 10min. After the removal of red blood cells, the cell pellet was re-suspended in PBS to produce single cell suspension.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eFlow cytometry\u003c/h2\u003e\u003cp\u003eIsolated primary single cell or RAW264.7 cells were incubated with TruStain FcX\u0026trade; PLUS for 10 minutes on ice to block Fc receptors. Subsequently, the cells were resuspended in PBS containing Zombie dyes solution and incubated at room temperature in dark for 20 minutes. For cell-surface staining, primary antibodies (mouse CD45, mouse F4/80, and mouse CD11c) were applied at their predetermined optimal concentrations, and the cells were incubated on ice for 20 minutes in the dark. Next, cells were incubated with the cyto-fast\u0026trade; Fix/Perm solution at room temperature and protected from light for 20 minutes. Following this, mouse CD206 staining was performed at room temperature for 20 minutes, Finally, the stained cells were resuspended in PBS and analyzed by flow cytometry (Thermo Fisher Scientific, Waltham, MA, USA). M1-like cells macrophages were defined as CD45\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e. M2-like macrophages were characterized as CD45\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e\u0026minus;\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e defined. (all reagents and antibodies used in this study were sourced from BioLegend (San Diego, CA), antibodies used were listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eCo-culture tumor cells with macrophage RAW264.7\u003c/h2\u003e\u003cp\u003eTranswell co-culture system (Corning, Glendale, AZ, USA): CAP1 knockdowns tumor cells were seeded in the upper chamber, while RAW264.7 macrophages were cultured in the lower chamber. After 48 h of co-culture, RAW264. 7 cells were collected for immunophenotyping analysis using flow Cytometry.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eConditioned medium induce Macrophage polarization\u003c/h2\u003e\u003cp\u003eObtain Conditioned medium (CM): CAP1 knockdowns tumor cells were cultured in T-175 flasks with complete medium until reaching 80% confluence. The medium was replaced with RPMI-1640 containing 1% fetal bovine serum, and cells were incubated for 48 h at 37\u0026deg;C in a 5% CO2 humidified atmosphere. Collect the supernatant, centrifuged at 1,000 \u0026times; g for 10 min to remove cellular debris, the conditioned medium (CM) was obtained by mixing supernatant and fresh complete medium 1:1 (v/v). For macrophage stimulation, RAW264.7 cells were treated with the prepared CM for specified durations under standard culture conditions. Macrophage polarization was detected by Flow Cytometry.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eRRM2 inhibitor recovery assay\u003c/h2\u003e\u003cp\u003e(1) in vitro: transwell migration assays were performed using 0.4 \u0026micro;m pore-size chambers (Corning, Glendale, AZ, USA). MC38 or CT26 cells transfected with shRNA plasmids were seeded in the upper chamber, while RAW264.7 macrophages were placed in the lower chamber. Tumor cells in the upper chamber were treated with RRM2 inhibitor at a concentration of 823 \u0026micro;M. Following 48 hours of co-culture, RAW264.7 macrophages were harvested for subsequent staining and flow cytometry analysis. (2) in vivo: Briefly, MC38 cells (4 \u0026times; 10⁶ cells in 100 \u0026micro;L PBS) were intraperitoneally injected into 6-week-old male C57BL/6 mice. The experimental groups received RRM2 inhibitors at doses of 100 mg/kg or 50 mg/kg every other day, while the control group was administered an equivalent volume of DMSO. Approximately 15 days post-inoculation, tumour-bearing mice were euthanized to harvest peritoneal tumors.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using GraphPad Prism 8 software (GraphPad Software, Inc., USA). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of biological replicate. Statistical significance was defined as *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for all measured parameters.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eCAP1 is downregulated in colorectal cancer and inhibits tumor growth in vivo\u003c/h2\u003e\u003cp\u003eTo elucidate the role of CAP1 in colorectal cancer (CRC) progression, we first compared CAP1 transcript expression in human CRC tissues and adjacent normal tissues using the TCGA database (The Cancer Genome Atlas Program). This reveals significantly downregulated CAP1 expression in CRC tissues (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, 1A). Moreover, higher CAP1 expression positively correlated with patient survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Consistent with these findings, WB and immunohistochemical staining of clinical samples demonstrated reduced CAP1 expression in tumour tissues compared to adjacent normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further investigate the functions of CAP1, we silenced Cap1 expression in mouse CRC cell lines (MC38 and CT26) using shRNA-encoding lentivirus. Successful knockdown was confirmed in both cell lines using two different shRNA designs (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB\u0026ndash;D). Interestingly, CAP1 downregulation did not alter cell proliferation rates although it significantly enhanced cell migration in vitro (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE\u0026ndash;G). To understand how CAP1 knockdown affects tumor growth in vivo, To validate our findings in vivo, we established subcutaneous mouse models by injecting shNC shCAP1-1, shCAP1-2 MC38 cells in immuno-competent C57BL/6 mice (n\u0026thinsp;=\u0026thinsp;6 per group) and monitored tumor size over 10 days. In difference with the trend observed in vitro, CAP1 knockdown markedly promoted tumor growth in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u0026ndash;G). Similarly, when intraperitoneally injected (n\u0026thinsp;=\u0026thinsp;5 per group), CAP1-knockdown MC38 cells grew much more quickly than control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, I). The lack of correlation between in vitro and in vivo experiments led us to suspect of the implication of tumor microenvironment. To test this hypothesis, we assessed tumor progression in immuno-deficient nude mice by subcutaneously injecting shNC shCAP1-1, shCAP1-2 MC38 cells (n\u0026thinsp;=\u0026thinsp;6 per group). Interestingly, CAP1 knockdown did not affect tumor growth (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eH\u0026ndash;J). In total, these results indicate that CAP1 is a tumor-suppressor, and its effect is mediated by the tumor immune microenvironment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eCAP1 silencing in colorectal cancer cells promotes macrophage M2-type polarization\u003c/h2\u003e\u003cp\u003eThe tumor immune microenvironment critically regulates colorectal cancer progression. Given our earlier finding that CAP1 knockdown accelerated CRC growth specifically in immuno-competent mice, we hypothesized that CAP1 modulates anti-tumor immunity. To test this hypothesis, we employed a peritoneal carcinomatosis model that permits facile evaluation of the tumor-immune crosstalk. Briefly, MC38 cells with CAP1 knockdown (shCAP1) and control (shNC) were intraperitoneal injected into C57BL/6 mice (n\u0026thinsp;=\u0026thinsp;6 per group). Subsequently, we isolated epididymal adipose tissue macrophages\u0026mdash;key immune sentinels in the peritoneal cavity and characterized their phenotypes using well-characterized surface biomarkers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, Fig.\u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). The results of the pre-experiment revealed that the proportion of macrophages changed significantly on the third day after intraperitoneal injection of tumour cells(Fig.\u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB-D). Interestingly, CAP1 silencing significantly enhanced macrophage polarization towards the immuno-suppressive M2 phenotype but not the M1 phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C, S2E). In addition to the peritoneal model, we employed the subcutaneous tumor model to examine macrophage infiltration, the results of which show markedly enhanced infiltration of CD163⁺ M2 macrophages in CAP1-knockdown tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo dissect the underlying mechanism, we used the transwell co-culture system to distinguishing contact-dependent from soluble-factor-mediated effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). This co-culture model physically separated tumor cells (in the upper chamber) from RAW264.7 macrophages (in the lower chamber). In line with the in vivo observation, CAP1-deficient CRC cells robustly induced M2 macrophage polarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-I). Crucially, this effect was recapitulated when macrophages were exposed to the conditioned media of CAP1-knockdown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ-M), demonstrating that soluble factors alone are sufficient to drive M2 macrophage polarization. Collectively, these data establish that CAP1 loss in CRC cells affected M2 macrophage polarization through secreting soluble factors.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCAP1 modulates M2 polarization through RRM2 upregulation and secretion.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHaving established that CAP1-deficient CRC cells secrete soluble factors driving M2 macrophage polarization, we next sought to identify the specific molecular mediator. Quantitative proteomic profiling of CAP1-knockdown MC38 cells revealed 817 differentially expressed proteins (216 upregulated, 601 downregulated) compared to control MC38 cells (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA-B), with further pathway enrichment implicating metabolic reprogramming and immune modulation (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC-D). Strikingly, ribonucleotide reductase regulatory subunit M2 (RRM2) emerged as one of the most significantly upregulated proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). Further analysis of the TCGA database uncovers elevated RRM2 transcripts in human CRC tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, we measured RRM2 protein levels in the cells and conditioned media using western blot and ELISA approaches, respectively. It was shown that CAP1-knockdown led to elevation RRM2 protein in MC38 and CT26 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) as well as in the conditioned media (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). To understand the effects of RRM2, we treated RAW264.7 macrophages with recombinant murine RRM2 (rRRM2) for 48h. It was found that rRRM2 induced M2 macrophage polarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), recapitulating the effect observed with the secretome of CAP1-knockdown cells. To validate these observations in vivo, we detected the expression of relevant molecules using animal tumor tissue. We found that in the tumors with low expression of CAP1, the expression of RRM2 increased and the number of M2-type macrophages increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Generally, these results indicate that CAP1 knockdown enhances the secretion of RRM2 which drives M2 macrophage polarization.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eAlkbh5-mediated m6A modification is involved in the regulation of RRM2 expression by CAP1\u003c/h2\u003e\u003cp\u003eOur previous findings established that CAP1 depletion in CRC cells promotes M2 macrophage polarization through upregulation of RRM2 protein. To elucidate the molecular mechanism underlying this regulation, we first confirmed that while RRM2 protein levels increased in CAP1-knockdown cells, quantitative PCR analysis does not show corresponding change in Rrm2 mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), effectively excluding transcriptional regulation as the primary mechanism. This observation led us to systematically investigate alternative regulatory pathways. Initial examination of protein stability using cycloheximide chase assays reveals unchanged RRM2 degradation kinetics in CAP1-deficient cells (Fig.\u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e), directing our attention toward post-transcriptional regulatory mechanisms involving RNA modifications that influence translation efficiency.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe hypothesized that CAP1 might regulate RRM2 through m6A-dependent translational control. This hypothesis was initially supported by our observation of significantly elevated global m6A levels in CAP1-knockdown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Further analysis of the previous proteomics data revealed that in CAP1-knockdown cells, the expression level of ALKBH5, an eraser of m6A modification, decreased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The mRNA expression level of ALKBH5 was decreased in cells with cap1 low-expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Moreover, overexpression of ALKBH5 significantly reduced the protein expression of RRM2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Together, these results provide evidence that CAP1 regulates RRM2 protein levels through ALKBH5-dependent m6A modification pathway.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eThe RRM2 inhibitor osalmid rescues CAP1-loss-driven tumor growth\u003c/h2\u003e\u003cp\u003eHaving established RRM2 as the mechanistic mediator between CAP1 loss and M2 macrophage polarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e), we next investigated whether pharmacological inhibition of RRM2 could reverse this effect. Osalmid was previously reported to inhibit RRM2\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Here, we first tested whether osalmid could inhibit M2 macrophage polarization and found that it significantly attenuated IL-4/IL-10-induced M2 polarization of RAW264.7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). Next, we tested the efficacy of osalmid to reduce M2 macrophage polarization induced by CAP1-knockdown CRC cells. In the transwell co-culture system, osalmid added to the lower chamber reduced the percentage of CD11b-CD206\u0026thinsp;+\u0026thinsp;cells to the basal level in MC38 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D) and CT26 cells (Fig. S5A-B), demonstrating the therapeutic potential of inhibiting RRM2.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurthermore, we investigated the efficacy of osalmid in the peritoneal carcinomatosis model. The mice were randomly divided into 6 groups (n\u0026thinsp;=\u0026thinsp;6 per group), including the control group, the CAP1 low-expression group, and the osalmid treatment group. Importantly, osalmid selectively inhibited growth of CAP1-knockdown tumors while showing no efficacy against control tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F). A similar trend was observed in the analysis of tumor-associated macrophages phenotypes, which demonstrates a sharp decline in M2 macrophage polarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-H). In conclusion, these results support that osalmid selectively inhibited the growth of CAP1-knockdown cells through reversing the immuno-suppressive phenotype of tumor-associated macrophages.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eColorectal cancer has long posed a significant threat to human health and life, although immunotherapy combined with surgical resection and chemoradiotherapy has significantly improved patients survival, the outcomes for some patients remain suboptimal. Therefore, it is urgent to find new therapeutic targets. In this study, bioinformatics analysis revealed that CAP1 is markedly downregulated in tumors and exhibits a significant positive correlation with patient survival, indicating its potential functional involvement in the pathogenesis and progression of colorectal cancer. In recent years, many studies have found that CAP1 is highly associated with cancer, such as: breast cancer, liver cancer, lung cancer, etc. In breast cancer (BC), CAP1 is highly expressed and significantly correlates with BC grade, and promotes BC cell proliferation and migration through down-regulation of C-terminal Binding Protein 2 (CtBP2) and E-cadherin expression\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. In addition, CAP1 is overexpressed in ovarian cancer (OC) and contributes to the cell cycle by affecting the cell cycle\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Although some studies suggest that CAP1 contributes to the development of various tumors, its role in colorectal cancer (CRC) remains less explored. Researchers have observed that CAP1 expression does not markedly rise in several CRC cell lines. Furthermore, CAP1 knockdown significantly boosts the migration of CRC cells without significantly affecting their proliferation\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Our results are consistent with these observations, as we too found that CAP1 knockdown markedly enhances CRC cell migration but has no significant impact on proliferation in CRC cell lines. However, in vivo experiments have shown that the absence of CAP1 can significantly promote tumor growth in immunocompetent mice, suggesting that CAP1's effect on CRC growth may be related to the tumor's immune microenvironment.\u003c/p\u003e\u003cp\u003eCancer is a group of diseases characterized by uncontrolled cell proliferation. In traditional tumor research, scientists focus on the effect of the intrinsic changes of cancer cells on tumor proliferation, migration, drug resistance, etc. However, there is increasing evidence showed that tumor cells reside in a complex microenvironment with diverse components, there are extensive interactions between cancer cells and the tumor microenvironment, and it is necessary to consider the tumor and its microenvironment as an integrated whole\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. The interplay between tumor cells and immune cells in the microenvironment plays an important role in tumor growth. Studies have indicated that tumor cells upregulate the expression of PMVK, to stabilize GAD1 and recruit ACAT1, resulting in elevated levels of 4-Ac-GABA in the tumor microenvironment. The 4-Ac-GABA binds to GABAAR on CD8\u003csup\u003e+\u003c/sup\u003e T cells, inhibiting the AKT1 signal pathway, which in turn suppresses the activation of CD8\u003csup\u003e+\u003c/sup\u003e T cells and the anti-tumor response\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Our findings revealed that CAP1 markedly suppressed the polarization of macrophages towards the M2 phenotype, irrespective of whether the macrophages were isolated in vivo or co-cultured with tumour cells in vitro.\u003c/p\u003e\u003cp\u003eN6-methyladenosine (m6A) RNA modification has emerged as a pivotal regulator in tumorigenesis and cancer progression. Accumulating evidence indicates that m6A plays a dual role in cancer, acting as either an oncogene or a tumor suppressor depending on the context\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. For instance, elevated expression of certain m6A methyltransferases, such as ALKBH5 inhibited Gastric cancer invasion and migration by modulating the stability of PKMYT1 mRNAs\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Moreover, m6A is intricately involved in shaping the tumor immune microenvironment\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. It can influence the infiltration and function of immune cells, thereby affecting tumor immune evasion. Our research results indicate that Alkbh5-mediated m6A modification of RRM2 mRNA significantly enhances the protein expression of RRM2 in CRC. RRM2 is associated with Ferroptosis in macrophages\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e, and promote polarization of macrophage to M2 macrophage\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Our research further substantiates that RRM2 can drive the polarization of macrophages towards the M2 phenotype. Moreover, osalmid, an inhibitor of RRM2, can significantly suppress the growth of colorectal cancer (CRC). These results highlight RRM2 inhibitors exhibit potent anticancer effects in CRC and RRM2 hold promise as potential therapeutic target for treating CRC.\u003c/p\u003e\u003cp\u003eThe present study uncovered a novel mechanism of action of CAP1 acting in RRM2 proteins, which is a novel finding, thus elucidating the mechanism by which CAP1 plays a role in colorectal cancer. Notably, the CAP1/RRM2 axis can be synergistically inhibited by conventional chemotherapeutic drug combinations that can inhibit CRC progression. In conclusion, we demonstrated that the CAP1/RRM2 axis in CRC cells regulates anti-tumor immunity through polarized macrophage M2 type. Our findings may provide new strategies for the clinical treatment of colorectal cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eEthics approval\u003c/h2\u003e\u003cp\u003eHuman study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Fourth People's Hospital of Chong qing (Chongqing Emergency Medical Center) (Approval Number: 2025 (54)). All animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of Chongqing University Central Hospital (Approval Number: 2412004)\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003cp\u003eInformed consent was obtained from all individual participants included in the study\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the Science and Technology Research Program of Chongqing Municipal Education Commission (Grant No. KJQN202400128), Chongqing Municipal Health Commission (Grant No 2024GDRC011) and Chongqing University (Grant No 2023CDJYGRH-YB10).\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\u003cp\u003eTianlin Feng: implementation of the research plan, participation in data analysis and paper writing. Ling Lin: experimental design, paper writing. Xiaoya Zhou and Li Li: cell experiments. Yao Chen and Wenyi Zheng: sample collection and processing. Qinrui Cai and Dongling Li: Data analysis and statistics. Jianshan Lin and Qianyao Wang: Literature review and background material organization. Wei Li and Xiaoyuan Zheng: experimental design and data analysis, providing professional opinions and technical support. Fan Yang: project initiation, research planning and design. All authors read and approved the final manuscript\u003c/p\u003e\u003ch2\u003eData Availability Statement\u003c/h2\u003e\u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. The MS proteomics data have been deposited at the iProX database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://iprox.org\u003c/span\u003e\u003cspan address=\"https://iprox.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with the program ID: PXD065883\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHardcastle JD, Thomas WM, Chamberlain J et al (1989) Randomised, controlled trial of faecal occult blood screening for colorectal cancer. Results for first 107,349 subjects. Lancet 1(8648):1160\u0026ndash;1164\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Giaquinto AN (2024) Jemal Cancer statistics, 2024. CA Cancer J Clin 74(1):12\u0026ndash;49\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBray F, Laversanne M, Sung H et al (2024) Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 74(3):229\u0026ndash;263\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOno S (2013) The role of cyclase-associated protein in regulating actin filament dynamics - more than a monomer-sequestration factor. J Cell Sci 126(Pt 15):3249\u0026ndash;3258\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJansen S, Collins A, Golden L et al (2014) Structure and mechanism of mouse cyclase-associated protein (CAP1) in regulating actin dynamics. J Biol Chem 289(44):30732\u0026ndash;30742\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu G, Swiston J (1994) Young Comparison of human CAP and CAP2, homologs of the yeast adenylyl cyclase-associated proteins. J Cell Sci 107(Pt 6):1671\u0026ndash;1678\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXue B (2013) Robinson Guardians of the actin monomer. Eur J Cell Biol 92(10\u0026ndash;11):316\u0026ndash;332\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShekhar S, Chung J, Kondev J et al (2019) Synergy between Cyclase-associated protein and Cofilin accelerates actin filament depolymerization by two orders of magnitude. Nat Commun 10(1):5319\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBergqvist M, Elebro K, Sandsveden M et al (2020) Effects of tumor-specific CAP1 expression and body constitution on clinical outcomes in patients with early breast cancer. Breast Cancer Res, 22(1)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRamsey A, Akana L, Miyajima E et al (2023) CAP1 (cyclase-associated protein 1) mediates the cyclic AMP signals that activate Rap1 in stimulating matrix adhesion of colon cancer cells. Cell Signal 104:110589\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee S, Lee HC, Kwon YW et al (2014) Adenylyl cyclase-associated protein 1 is a receptor for human resistin and mediates inflammatory actions of human monocytes. Cell Metab 19(3):484\u0026ndash;497\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhu Y, Wan N, Shan X et al (2021) Celastrol targets adenylyl cyclase-associated protein 1 to reduce macrophages-mediated inflammation and ameliorates high fat diet-induced metabolic syndrome in mice. Acta Pharm Sin B 11(5):1200\u0026ndash;1212\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXiao Y (2021) Yu Tumor microenvironment as a therapeutic target in cancer. Pharmacol Ther 221:107753\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDi Martino JS, Mondal C (2019) Bravo-Cordero Textures of the tumour microenvironment. Essays Biochem 63(5):619\u0026ndash;629\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOsipov A, Murphy A (2019) Zheng From immune checkpoints to vaccines: The past, present and future of cancer immunotherapy. Adv Cancer Res 143:63\u0026ndash;144\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCassetta L (2018) Pollard Targeting macrophages: therapeutic approaches in cancer. Nat Rev Drug Discov 17(12):887\u0026ndash;904\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDeNardo DG (2019) Ruffell Macrophages as regulators of tumour immunity and immunotherapy. Nat Rev Immunol 19(6):369\u0026ndash;382\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWatanabe S, Alexander M, Misharin AV et al (2019) The role of macrophages in the resolution of inflammation. J Clin Invest 129(7):2619\u0026ndash;2628\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYeung OW, Lo CM, Ling CC et al (2015) Alternatively activated (M2) macrophages promote tumour growth and invasiveness in hepatocellular carcinoma. J Hepatol 62(3):607\u0026ndash;616\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang N, Liang H (2014) Zen Molecular mechanisms that influence the macrophage m1-m2 polarization balance. Front Immunol 5:614\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBoutilier AJ (2021) and S.F. Elsawa Macrophage Polarization States in the Tumor Microenvironment. Int J Mol Sci, 22(13)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZuo Z, Zhou Z, Chang Y et al (2024) Ribonucleotide reductase M2 (RRM2): Regulation, function and targeting strategy in human cancer. Genes Dis 11(1):218\u0026ndash;233\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eD'Angiolella V, Donato V, Forrester FM et al (2012) Cyclin F-mediated degradation of ribonucleotide reductase M2 controls genome integrity and DNA repair. Cell 149(5):1023\u0026ndash;1034\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXiong W, Zhang B, Yu H et al (2021) RRM2 Regulates Sensitivity to Sunitinib and PD-1 Blockade in Renal Cancer by Stabilizing ANXA1 and Activating the AKT Pathway. Adv Sci (Weinh) 8(18):e2100881\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTang B, Xu W, Wang Y et al (2021) Identification of critical ferroptosis regulators in lung adenocarcinoma that RRM2 facilitates tumor immune infiltration by inhibiting ferroptotic death. Clin Immunol, 232\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTang B, Xu W, Wang Y et al (2021) Identification of critical ferroptosis regulators in lung adenocarcinoma that RRM2 facilitates tumor immune infiltration by inhibiting ferroptotic death. Clin Immunol 232:108872\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTang Q, Wu L, Xu M et al (2020) Osalmid, a Novel Identified RRM2 Inhibitor, Enhances Radiosensitivity of Esophageal Cancer. Int J Radiat Oncol Biol Phys 108(5):1368\u0026ndash;1379\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu X, Yao N, Qian J et al (2014) High expression and prognostic role of CAP1 and CtBP2 in breast carcinoma: associated with E-cadherin and cell proliferation. Med Oncol 31(3):878\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu XF, Ni QC, Chen JP et al (2014) Knocking down the expression of adenylate cyclase-associated protein 1 inhibits the proliferation and migration of breast cancer cells. Exp Mol Pathol 96(2):188\u0026ndash;194\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHua M, Yan S, Deng Y et al (2015) CAP1 is overexpressed in human epithelial ovarian cancer and promotes cell proliferation. Int J Mol Med 35(4):941\u0026ndash;949\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhou X, Chen Z, Yu Y et al (2024) Increases in 4-Acetaminobutyric Acid Generated by Phosphomevalonate Kinase Suppress CD8(+) T Cell Activation and Allow Tumor Immune Escape. Adv Sci (Weinh) 11(43):e2403629\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang T, Kong S, Tao M et al (2020) The potential role of RNA N6-methyladenosine in Cancer progression. Mol Cancer 19(1):88\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHu Y, Gong C, Li Z et al (2022) Demethylase ALKBH5 suppresses invasion of gastric cancer via PKMYT1 m6A modification. Mol Cancer, 21(1)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao L, Li Q, Zhou T et al (2024) Role of N6-methyladenosine in tumor neovascularization. Cell Death Dis 15(8):563\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTang B, Zhu J, Wang Y et al (2023) Targeted xCT-mediated Ferroptosis and Protumoral Polarization of Macrophages Is Effective against HCC and Enhances the Efficacy of the Anti-PD-1/L1 Response. Adv Sci (Weinh) 10(2):e2203973\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStatements \u0026amp; Declarations\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":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"colorectal cancer, CAP1, RRM2, m6A, Macrophage Polarization","lastPublishedDoi":"10.21203/rs.3.rs-7355735/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7355735/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eColorectal cancer (CRC) progression is critically regulated by dynamic interactions between tumor cells and tumor-associated macrophages (TAMs), which shape the immuno-suppressive tumor microenvironment. In this study, we identify CAP1 as a novel regulator of this crosstalk through its control of RNA methylation-dependent M2 macrophage polarization. Clinical analysis reveals significant CAP1 downregulation in CRC tissues compared to adjacent normal mucosa, with its expression positively correlating with patient survival outcomes. While CAP1 knockdown did not show cell-autonomous effects on CRC proliferation in vitro, it dramatically enhanced tumor growth in immuno-competent mouse models. Further mechanistic studies uncover that CAP1 deficiency in tumor cells triggers a ALKBH5-mediated increase in m⁶A RNA methylation, specifically enhancing the translation and secretion of RRM2. This tumor-derived RRM2 potently drives M2 polarization of TAMs, creating a pro-tumorigenic niche that facilitated the proliferation of CRC cells in turn. Moreover, the RRM2 inhibitor shows considerable efficacy in treating tumors with low expression of CAP1. Collectively, this study provides new insights into how tumor cells regulate immune responses through post-transcriptional modification and suggests potential therapeutic strategies targeting the CAP1-RRM2 axis in CAP1-deficient CRCs.\u003c/p\u003e","manuscriptTitle":"CAP1-Mediated m6A Modification of RRM2 Suppresses Tumor-Associated M2 Macrophage Polarization and Colorectal Cancer Growth","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-26 12:10:45","doi":"10.21203/rs.3.rs-7355735/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2025-10-17T21:37:12+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-10-01T05:42:30+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-17T13:00:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-16T13:45:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2025-08-14T21:39:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3de93b82-de6a-4fac-a30c-3122adaa1e32","owner":[],"postedDate":"September 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-05T15:58:58+00:00","versionOfRecord":{"articleIdentity":"rs-7355735","link":"https://doi.org/10.1007/s00018-025-06031-x","journal":{"identity":"cellular-and-molecular-life-sciences","isVorOnly":false,"title":"Cellular and Molecular Life Sciences"},"publishedOn":"2026-01-02 15:57:01","publishedOnDateReadable":"January 2nd, 2026"},"versionCreatedAt":"2025-09-26 12:10:45","video":"","vorDoi":"10.1007/s00018-025-06031-x","vorDoiUrl":"https://doi.org/10.1007/s00018-025-06031-x","workflowStages":[]},"version":"v1","identity":"rs-7355735","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7355735","identity":"rs-7355735","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}