ONECUT2 governs the POU6F2-beta-catenin axis to modulate cancer stemness and drives the CCL28-dependent pathway for macrophage polarization in breast cancer

ONECUT2 governs the POU6F2-beta-catenin axis to modulate cancer stemness and drives the CCL28-dependent pathway for macrophage polarization in breast cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article ONECUT2 governs the POU6F2-beta-catenin axis to modulate cancer stemness and drives the CCL28-dependent pathway for macrophage polarization in breast cancer Xiubao Ren, Meng Shen, Haixia Jin, Ziqi Huang, Nan Dong, Gen Liu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7518380/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Cancer stem cells (CSC) and the immunosuppressive microenvironments are key drivers of breast cancer (BC) progression and drug resistance. However, the molecular mechanisms by which One Cut Homeobox 2 (ONECUT2) governs CSC and the tumor immune microenvironment (TIME) remains largely unknown. Given the critical knowledge gap, we sought to investigate ONECUT2’s regulatory impact on CSC properties and TIME profiles using breast cancer cell lines, animal models, and clinical specimens. Here, we demonstrated that ONECUT2, a core transcription factor, mediates CSC characteristics and reprograms the TIME to drive macrophage polarization to the M2-type, a tumor-promoting state. Mechanistically, ONECUT2 inhibition transcriptionally activated POU6F2, which subsequently triggered beta-catenin, thereby enhancing CSC properties and chemoresistance in BC. With respect to modulating the immune microenvironment, ONECUT2 can govern macrophage polarization, identifying CCL28 as a transcriptional target of ONECUT2 required for M2-type macrophage polarization, and CCR10 as a key receptor involved in immune modulation. These findings highlight the critical involvement of ONECUT2 in modulating BC stemness via targeting the POU6F2-beta-catenin axis and managing macrophage polarization to M2 phenotype through the CCL28-CCR10 pathway. Our study suggests that ONECUT2 modulates cancer stemness and the immune microenvironment, and that targeting it along its downstream axis may provide an effective approach for BC treatment. Health sciences/Diseases/Cancer/Breast cancer Health sciences/Diseases/Cancer/Cancer microenvironment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Breast cancer (BC) is the predominant malignant tumor among women and has a profound impact on their health [1]. Cancer stem cells (CSC) exhibit intrinsic resistance to chemotherapeutic agents, which contributes to tumor progression and recurrence in BC. Exploring how chemotherapy regulates BC stemness is of great significance for reversing chemoresistance and ultimately improving patient prognosis. In our previous study [2] , we identified a novel mechanism by which chemotherapy induces BC cells to secrete extracellular vesicles (EVs) that serve as critical mediators of therapeutic resistance. These EVs targeted the transcription factor One Cut Homeobox 2 (ONECUT2), thereby inducing a CSC-like phenotype and chemoresistance in BC. ONECUT2, a core member of the ONECUT family, is a transcription factor that is involved in multidirectional differentiation [3]. ONECUT2 plays a role in various oncogenic processes, including tumor proliferation, metastasis, epithelial-mesenchymal transition (EMT), angiogenic signaling, resistance to endocrine therapy, and subtype switching in BC [4-6]. However, the effects of ONECUT2 on CSC properties are complex and involve interactions with multiple signaling pathways and molecular mechanisms that collectively reshape CSC phenotypes. As a transcription factor, ONECUT2 modulates CSC traits by regulating the downstream genes PPP2R4 [7] or TFPI [8] in gastric cancer (GC). Another study [4] elucidates the roles of ONECUT2 in stemness maintenance, verifying beta-catenin as a crucial downstream effector. In non-small cell lung cancer (NSCLC) [9], ONECUT2 has emerged as a vital downstream effector in CSC driven by lncRNA THUMPD3-AS1, highlighting its potential as a therapeutic target in NSCLC. Although our findings suggest a regulatory role of ONECUT2 in CSC properties, the underlying molecular mechanisms are poorly understood. This knowledge gap underscores the need to fully elucidate the mechanism by which ONECUT2 mediates functional plasticity in CSC, thereby paving the way for the development of novel therapeutic strategies. In our study, we found that ONECUT2 transcriptionally targets POU6F2 (POU domain, class 6, transcription factor 2), thereby triggering beta-catenin and modulating cancer stemness and chemoresistance in BC cell lines, animal models, and clinical specimens. Emerging evidence indicates that the interplay between CSC and the tumor immune microenvironment (TIME) drives tumor progression and immune evasion [10-13] . In NSCLC, ONECUT2 serves as a master mediator of the exhausted CD8 + T cell phenotype [14]; thus, it has been identified as a critical regulator of cytotoxic immune responses and a novel therapeutic target for checkpoint inhibition. Another study identified ONECUT2 as a direct target of T-bet [15], establishing a central role in the differentiation of T-helper (Th) progenitor cells to Th1/Th2 effector cells. However, the precise role of ONECUT2 in the BC TIME remains unknown. Here, we observed through in vivo and in vitro experiments that ONECUT2 functions as a key regulator of M2-type macrophage polarization, which reprograms the TIME. This study further elucidates the regulatory network of the immunosuppressive microenvironment and screens for potential therapeutic targets to enhance the efficacy of immunotherapy in BC. Materials and Methods Cells and constructs The human BC cell lines (MDA-231, MCF-7 and BT474), mouse BC cell line (4T1) and human THP-1 cell line were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in the recommended media. All cancer cell lines have been authenticated by STR genotyping and proved to be free of mycoplasma contamination. MDA-231 cells with stable ONECUT2 overexpression (MDA-231/OC-2) were generated by ONECUT2 overexpression plasmid (OriGene; Rockville, MD), and subsequently selected by G418 solution. 4T1 cells with stable ONECUT2 overexpression (4T1/OC-2) were generated by transduction with lentivirus carrying pLVX-Onecut2 (mouse, NM_194268.3), followed by puromycin selection, and control cells were generated using pLVX empty vector. The siRNAs targeting ONECUT2, POU6F2, and CTNNB1, along with negative control siRNA, were purchased from Qiagen (Venlo, Netherlands). RNA or DNA was transfected using Lipofectamine RNAiMAX (Invitrogen; Carlsbad, CA) or Lipofectamine 3000 (Invitrogen; Carlsbad, CA) following the manufacturer’s protocols. Patients and tissue samples Tissue microarrays (TMAs) comprising 99 BC samples coupled with patients’ survival data were acquired from Shanghai Outdo Biotech (Shanghai, China). All experimental procedures were conducted following approval by the Ethics Committee of Tianjin Medical University Cancer Institute and Hospital (E2021075) and conducted in accordance with the Declaration of Helsinki. The clinical parameters of the patients were shown in Table S1. Real-time quantitative PCR and Western blotting RNA extraction by TRIZOL (Invitrogen; Carlsbad, CA) and real-time quantitative PCR (RT-qPCR) were performed as previously described [2], with β-ACTIN as a control (primer sequences in Table S2). Proteins were extracted using RIPA lysis buffer, with antibodies listed in Table S2. Nuclear proteins were extracted using the Nuclear Protein Extraction Kit (Bestbio; Shanghai, China) following the manufacturer’s protocol. Sphere formation assay Treated MCF-7 cells were harvested and re-suspended in serum-free medium. The single-cell suspension was diluted to a density of 2 000 cells per well and seeded into ultralow attachment 6-well plates. The number of spheres was counted on day 14, and the sphere-forming efficiency was calculated using a diameter ≥70μm as the criterion. Enzyme-linked immunosorbent assay Cultured supernatants were collected and assayed for enzyme-linked immunosorbent assay (ELISA) by ElaBoXTMHuman MEC (CCL28) ELISA Kit (Solarbio; Beijing, China) following the manufacturer’s instructions. Flow cytometry Single-cell suspensions were prepared from tumor tissues/spleens of mice or treated BC cells, followed by staining with antibodies (listed in Table S2) for flow cytometry (FCM) analyses. All samples were analyzed using a FACS with 3 replicates, and at least 10 000 events were acquired for each sample. Dual-luciferase reporter assay MCF-7 cells were seeded at about 60% confluence in 24-well plates. For promoter reporters, 0.5μg pGL3-Basic luciferase reporters were transfected into MCF-7 cells with ONECUT2 or POU6F2 overexpression. Empty pCMV6-Entry or pcDNA3.1(+) vector were used as control groups. The luciferase activities were determined using the Dual-Luciferase Reporter Assay System (Promega), as described previously [2]. Primer sequences are listed in Table S2. ChIP-PCR MCF-7 cells were harvested and performed using the ChIP Assay kit (Cell Signaling Technology, Massachusetts, USA.) under the manufacturer’s instructions. The DNA enrichment was assessed by real-time PCR using the specific primers (detailed in Table S2). Co-Immunoprecipitation The co-immunoprecipitation (Co-IP) assay was performed using the Epizyme Co-IP Kit (YJ201; Epizyme Biotech, China) according to the manufacturer’s instructions. In brief, lysates of 1×10 7 MDA-231 cells were incubated with anti-ONECUT2 antibody or IgG isotype control overnight at 4°C, subsequently co-incubated with protein A/G magnetic beads for 6 h, and further eluted for western blotting. Table S2 displays the used antibodies. Cell viability Cell viability was assessed using Cell Counting Kit-8 (CCK8, MedChemExpress, China). Briefly, MDA-231 cells were seeded at 2 500 cells/well using flat-bottomed 96-well culture plates and treated with docetaxel (DTX, 4 nM, Beyotime Biotechnology, China) or doxorubicin (DOXO, 125 nM, Sigma-Aldrich, USA). At indicated time points, 10μl CCK8 solution was added to each well followed by 2 h incubation at 37℃, and absorbance at 450 nm was measured. Immunohistochemistry (IHC) and multiplex IHC staining (mIHC) Formaldehyde-fixed, paraffin-embedded mouse tumor tissues was processed for IHC as previously described [16]. The antibodies were listed in Table S2. The stained slides were evaluated according to two criteria: 1) staining intensity (-: 0, +: 1, ++: 2, +++: 3) and 2) percentage of staining-positive tumor cells (0%: 0, 1~29%: 1, 30~69%: 2, ≥ 70%: 3). The intensity and percentage scores were multiplied to generate a final score, which were used in statistical analyses. mIHC staining was conducted by PANO 7-plex IHC Kit (Panovue, Beijing, China). DAPI was used for nuclear localization. The antibodies and their dilution ratios were listed in Table S2. Animals All animal experiments were approved by the Institutional Animal Care and Use Committee of Tianjin Medical University Cancer Institute and Hospital (E2021128). Six-week-old female NOD/SCID/IL2Rγ-null (NSG) mice and BALB/c mice were obtained from the Weitonglihua Corporation (Beijing, China). For the 4T1 mouse models, 4T1/OC-2 or 4T1 cells were injected into the mammary fat pad, and the tumor volume was measured using calipers. Tumors were harvested 20 days after injection. For the NSG mouse models, MDA-231/OC-2 or MDA-231 cells were injected into the mammary fat pads. Once the tumor volume reached ~300 mm 3 , the mice were divided into two groups: one group was sacrificed immediately (pre-DTX group), and the other group (post-DTX group) was treated with DTX (15 mg/kg) for three weeks and sacrificed three days after the final DTX treatment. Tumor tissues were cut into two pieces for subsequent experiments. Statistical analysis GraphPad Prism version 10.4.2 (GraphPad Software, USA) was used for graph drawing and statistical analysis. The results were derived from at least three independent experiments. Statistical significance between groups was assessed using Student's unpaired t-test, Student's paired t-test, Mann-Whitney test, one-way analysis of variance (ANOVA) for multiple comparisons, Pearson's correlation analysis, χ² test, log-rank test, over-representation analysis, and the Cox proportional hazards regression models, as appropriate. Statistical significance was set at p< 0.05. Results ONECUT2 regulates breast cancer stemness via the beta-catenin-dependent pathway Our previous study showed that ONECUT2 serves as a crucial determinant of CSC [2]; however, the underlying molecular mechanism remains poorly understood. Since the beta-catenin signaling pathway acts as a core set, with its target genes commonly serving as well-known stem-cell markers [17], we examined whether ONECUT2 could affect beta-catenin expression and clarify the regulatory mechanism. In MDA-231, MCF-7, and BT474 cell lines, ONECUT2 inhibition activated beta-catenin and upregulated stemness genes Notch1 and Nanog (Fig.1A). Conversely, ONECUT2 overexpression exhibited the opposite effect (Fig. 1B). We further observed a significant accumulation in nuclear beta-catenin rather than pGSK3β in BC cells (Fig. 1C), indicating that ONECUT2 activates the transcriptional activity of beta-catenin. However, this regulation did not directly bind to the promoter of beta-catenin, as evidenced by a luciferase assay showing that ONECUT2 had no effect on the CTNNB1 (encoding beta-catenin) promoter (Fig. S1). Beta-catenin attenuated the effect of ONECUT2 on the CD24 - CD44 + population known to be enriched in CSC (Fig.1D), sphere-forming efficiency (Fig. 1E, S2), and the expression of stemness gene Notch1 and Nanog (Fig. 1F) in BC cells. mIHC staining confirmed a significant inverse correlation (r=-0.2207, p=0.0281) between the expression of ONECUT2 and beta-catenin in BC patients (Fig. 1G, H). Disease-free survival (DFS) and overall survival (OS) were significantly shorter in the ONECUT2-low expression cohort (DFS, p< 0.0001; OS, p= 0.0455; Fig. I, Table 1). Collectively, these findings suggest that ONECUT2 exerts its CSC-modulatory role via a beta-catenin-dependent pathway. ONECUT2 regulates the transcriptional activity of POU6F2 The potential target genes of ONECUT2 were predicted using the Gene Transcription Regulation Database (GTRD) (http://gtrd.biouml.org/). When setting the transcription factor-binding site at the promoter [-2600, +100], we observed that POU6F2 was a promising target gene (Table S3). Given that POU6F2, reported to be regulated by ONECUT2 in embryonic retinas [3], was upregulated in ONECUT2-knockdown BC cells in our previous study [2], we conducted experiments to observe whether POU6F2 serves as a direct transcriptional target of ONECUT2, thereby mediating the regulatory effects of ONECUT2 on beta-catenin. In BC cells, ONECUT2 knockdown promoted POU6F2 expression (Fig. 2A), whereas ONECUT2 overexpression reduced POU6F2 expression (Fig. 2B). mIHC staining showed that ONECUT2 and POU6F2 exhibited a significant negative correlation (r=-0.2021, p=0.0449). DFS (p=0.0003) and OS (p=0.0031) were significantly prolonged in patients with low POU6F2 levels (Fig. 2C, Table 1). Further, we employed a Co-IP assay to confirm the binding among Onecut2, Pou6f2, and beta-catenin (Fig. 2D). Dual-luciferase reporter assays (Fig. 2E) and ChIP-PCR analysis (Fig. 2F) demonstrated that ONECUT2 regulates POU6F2 transcriptional activity and directly binds to its promoter region. These results provide conclusive evidence that POU6F2 is a direct transcriptional target of ONECUT2. POU6F2 promotes breast cancer stemness via activating beta-catenin Based on these findings, we speculated that POU6F2 may play a core role in exerting ONECUT2-mediated beta-catenin activation and CSC promotion in BC. POU6F2 deficiency in MDA-231, MCF-7, and BT474 cells resulted in decreased protein levels of beta-catenin and stemness-associated genes, including Notch1 and Nanog (Fig. 3A). Conversely, POU6F2 overexpression markedly increased the expression of these proteins (Fig. 3B), suggesting a regulatory function of POU6F2 on beta-catenin and the maintenance of CSC. Furthermore, the sphere formation ability (Fig. 3C) and the CD24 - CD44 + cell population (Fig. 3D) decreased with POU6F2 knockdown in MCF-7 cells. Following POU6F2 inhibition, we observed reduced resistance to DTX and DOXO in MDA-231 cells (Fig. 3E), underscoring POU6F2’s critical roles in promoting chemoresistance in BC cells. Furthermore, we observed a notable reduction of beta-catenin and stemness genes (Fig. 3F), the CD24 - CD44 + cell population (Fig. 1D), and the sphere formation ability (Fig. 1E) in the ONECUT2-deficient group with POU6F2 knockdown. As shown in Fig. 3G, POU6F2 demonstrated a strong positive correlation with beta-catenin (r=0.4496, p＜0.0001). Furthermore, patients with elevated beta-catenin expression exhibited significantly shortened DFS (p=0.0074), while no statistical association was observed between beta-catenin levels and OS (p=0.4479, Table 1). Mechanistically, using dual-luciferase reporter assays, we found that POU6F2 served as a transcriptional activator of CTNNB1, revealing significantly increased promoter activity upon POU6F2 overexpression (Fig. 3H). ChIP-PCR analysis further confirmed that POU6F2 binds to the promoter region of CTNNB1 (Fig. 3I). Taken together, these data highlight POU6F2 as a crucial mediator in activating beta-catenin, thereby promoting cancer stemness and chemoresistance in BC. The ONECUT2-POU6F2-beta-catenin axis enhances breast cancer stemness and chemoresistance in vivo To further evaluate the role of the ONECUT2-POU6F2-beta-catenin axis in modulating BC stemness and chemotherapy resistance, we constructed BALB/c mouse models bearing 4T1/OC-2 cells and 4T1 cells (Fig. 4A). A significant decrease of Pou6f2 and beta-catenin expression was observed in 4T1/OC-2 tumors relative to 4T1 tumors (Fig. 4B). Another NSG mouse model was generated, comprising MDA-231 cells and MDA-231/OC-2 cells (Fig. 4C), to analyze the role of the ONECUT2-POU6F2-beta-catenin axis in mediating CSC and chemoresistance in vivo. In MDA-231 tumor models, DTX treatment resulted in decreased Onecut2 expression, accompanied by increased expression of Pou6f2, beta-catenin, and Nanog (Fig. 4D, 4F), whereas it had no effect in MDA-231/OC-2 tumors (Fig. 4E). These results further support the critical role of the ONECUT2-POU6F2-beta-catenin axis in maintaining cancer stemness and chemoresistance in BC. ONECUT2 drives macrophage polarization to M2 phenotype in the breast cancer microenvironment Growing evidence suggests that there are complex interactions between CSCs and TIME [18, 19]. To determine the regulatory roles of ONECUT2 in the TIME, FCM was used to assess dynamic changes in immunocyte subsets within the tumor tissues and spleens of 4T1/OC-2 and 4T1 mice. We observed a notable reduction in M2 macrophage infiltration and an increase in the proportion of B cells in 4T1/OC-2 tumors (Fig. 5A, B). Spleen tissues from 4T1/OC-2 mice exhibited significant reductions in M2 macrophages and CD4 + T cell infiltration, accompanied by elevated percentages of CD3 + T cells, CD8 + T cells, B cells, and regulatory T cells (Tregs) (Fig. 5C, S3). Consistent results were obtained for THP-1-derived macrophages. ONECUT2 inhibition increased the CD163 + M2 macrophage population (Fig. 5D left, S4 upper panel) and the expression of the pro-cancer M2 markers CD163 and CD206 (Fig. 5E, left). Conversely, ONECUT2 overexpression demonstrated opposite effects (Fig. 5D right, S4 lower panel, and 5E right). As shown in Fig. 5F, silencing ONECUT2 increased the mRNA levels of IL-10 and Arg-1 produced by macrophages, whereas ONECUT2 overexpression was correlated with reduced mRNA levels of these genes. Our study indicated that ONECUT2 acts as a vital regulator of M2-type macrophage polarization in BC, underscoring its potential as a therapeutic target to reverse the immunosuppressive microenvironment and enhance the efficacy of immunotherapy. CCL28 is a direct target of ONECUT2 required for M2 macrophage polarization To further investigate the regulatory mechanism of ONECUT2 in M2 macrophage polarization, we analyzed the publicly available RNA-seq data from Irene et al. (GEO accession: GSE242541) [6] and observed that CCL28 was downregulated in MCF-7 and BT474 cells overexpressing ONECUT2 (Fig. 6A). Given that CCL28 [20] participates in BC progression by modulating proliferation, invasion, and metastasis, we investigated how ONECUT2 regulates CCL28 expression and subsequently reprograms macrophages toward the M2 phenotype. Decreased CCL28 expression in response to ONECUT2 overexpression was confirmed in BC cell lines at both the RNA (Fig. 6B, upper panel) and protein levels (Fig. 6C), whereas ONECUT2 deficiency produced the opposite effects (Fig. 6B lower panel, 6D). ELISA confirmed that ONECUT2 knockdown significantly elevated the secretion of CCL28 (Fig. S5). Furthermore, CCL28 expression was downregulated in both 4T1/OC-2 tumors (Fig. 6E) and MDA-231/OC-2 xenografts (Fig. 6F) compared with their respective controls (4T1 and MDA-231). Western blotting analysis showed that CCL28 overexpression partially rescued the effect of ONECUT2 on CCL28 expression in MDA-231 cells (Fig. 6G). Increased CCL28 expression led to an increase in the CD163 + M2 macrophage population and elevated expression of M2 markers (CD163 and CD206), which were partially blocked by ONECUT2 overexpression (Fig. 6H, S6A). Based on predictions from the JASPAR database, dual-luciferase reporter assays demonstrated that ONECUT2 significantly repressed the transcriptional activity of CCL28 in MCF-7 cells (Fig. 6I). ChIP-PCR further confirmed that ONECUT2 binds to the CCL28 promoter (Fig. 6J). Additionally, to investigate the receptors of CCL28 involved in the regulation of M2-type macrophage polarization, we examined the expression of CCR3 and CCR10 (known as CCL28 receptors). Knockdown of ONECUT2 in MDA-231 cells increased the RNA and protein levels of CCR10 in macrophages, whereas ONECUT2 overexpression induced the opposite effect. However, there were no significant changes in the CCR3 protein levels (Fig. 6K, 6L). The induction of CCR10 by CCL28 overexpression was reversed by ONECUT2 overexpression in MDA-231 cells (Fig. S6B). These findings suggest that CCR10 might act as a key molecule in ONECUT2-CCL28-mediated regulation of macrophage polarization to the M2 phenotype. Collectively, these results indicate that ONECUT2 transcriptionally targets CCL28, thereby driving macrophage polarization to an immunosuppressive M2-like phenotype. The ONECUT2-CCL28-CCR10 signaling axis reprograms macrophage polarization and modulates the TIME in BC. Discussion Here, we demonstrate a specific mechanism by which ONECUT2 regulates cancer stemness characteristics and affects the immunosuppressive environment in BC. Our current study reveals novel mechanisms underlying cancer cell self-renewal capacity and immunoregulatory function, which alleviate chemoresistance and improve survival outcomes in patients with BC. Growing evidence suggests that ONECUT2 is a multifaceted regulator of CSC properties and that its expression is significantly correlated with clinical prognosis [7, 21-23]. As shown in our previous study, BC-derived EVs promote CSC traits and chemotherapy resistance by downregulating ONECUT2 expression. Other studies [4, 9] further reveal that ONECUT2 is an effective regulator in maintaining cancer stemness and chemoresistance, while the precise mechanism remains to be fully elucidated. Recent studies have shown that ONECUT2 is a master regulator of CSCs properties through transcriptional reprogramming. ONECUT2 activates the transcriptional activity of TFPI in GC [8], thereby contributing to CSC traits and oxaliplatin resistance. Another study demonstrates that ONECUT2 inhibits PPP2R4 transcription [7], which in turn increases AKT/beta-catenin phosphorylation, thereby inducing GC stemness. Furthermore, ONECUT2 exerts a positive association with Wnt/beta-catenin signaling pathway activation [21], revealing a significant correlation between ONECUT2 expression and beta-catenin pathway activity. The beta-catenin signaling was reported as a core pathway in maintaining CSC phenotype, with its target genes commonly serving as established stem-cell markers [24]. Modulation of homeostasis facilitates stem-cell self-renewal balance [25]. In our current study, we identified beta-catenin functions as a central effector in ONECUT2-driven CSC maintenance and chemoresistance. ONECUT2 inhibition activated beta-catenin and induced a CSC phenotype in BC, suggesting beta-catenin as a pivotal target in ONECUT2-mediated cancer stemness property. POU6F2 was originally cloned from the human retina and is known as retina-derived POU-domain factor-1(RPF-1). The current understanding of POU6F2 is largely confined to the differentiation and morphogenesis of the kidney [26] and eye cornea [27]. Although several studies have elucidated the function of POU6F2 in tumor biology, its regulatory role in maintaining cancer stemness remains largely unexplored. POU6F2 exhibits dual regulatory roles in cancer, with tumor-promoting or tumor-suppressive functions influenced by microenvironmental factors across malignancies. In gastric adenocarcinoma with liver metastasis [28] and renal cell cancer [29], it functions as an oncogene, and upregulated expression is associated with a worse clinical prognosis. Conversely, POU6F2 exerts tumor-suppressive effects in Wilms’ tumor [30] and prolactinoma [31], which serve as potential targets for cancer treatment. In this study, we first demonstrated that ONECUT2 inhibition activates the transcriptional activity of POU6F2, subsequently inducing CSC phenotype and chemotherapy resistance through a beta-catenin-dependent pathway. These findings add a mechanism of mediating chemotherapy-driven BC stemness through the ONECUT2-POU6F2-beta-catenin axis, and suggest novel therapeutic targets for reversing chemoresistance. The immunosuppressive TIME is a major barrier to effective cancer immunotherapies. Reprogramming tumor-associated macrophages (TAMs) into an antitumor phenotype is a promising strategy. Studies [32, 33] have revealed that TAMs engage in functional crosstalk with the CSC phenotype. TAMs undergo preferential polarization toward the M2 phenotype, which acquires potent immunosuppressive activity that mediates pro-tumorigenic functions in BC [34]. In our study, we analyzed the changes in various immunocyte subsets in 4T1/OC-2 BALB/c mouse models and found a significant reduction in macrophage polarization towards the M2 type. Functionally, as evidenced by the RNA-Sequencing results shown in Fig. 6A, we figured out that CCL28 is a key mediator of ONECUT2-driven M2-type macrophage polarization. ONECUT2 knockdown leads to the transcriptional activation of CCL28, which subsequently induces macrophage polarization via the CCL28-CCR10 signaling axis, thereby creating an immunosuppressive TIME in BC. CCL28, a member of the CC chemokine subfamily, has been implicated in the promotion of tumor progression through immune regulation and EMT modulation [35, 36] . While a previous study [37] on colorectal cancer reported that CCL28 suppressed macrophage polarization to the M2 phenotype and inhibited tumor progression, our findings revealed a paradoxical pro-tumorigenic role in BC, where CCL28 upregulation enhanced M2-type polarization through a CCR10-dependent mechanism. This discrepancy may stem from the heterogeneity of the TIME and the presence of other mechanisms and factors that influence the interplay between CCL28-M2 macrophage, which requires further exploration. Our findings are the first to demonstrate the specific characteristics of ONECUT2 that shape transcriptional profiles linked to CSC traits and TIME in BC (Fig. 7). Conceivably, targeting pivotal molecules in the ONECUT2-POU6F2-beta-catenin and ONECUT2-CCL28-CCR10 signaling pathways could reduce cancer cell stemness as well as enhance the efficacy of chemotherapy and immunotherapy in BC. Declarations Conflict of Interest The authors declare no competing interests. Availability of Data and Materials The data generated in this study are available upon request from the corresponding author. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (No. 82103001, 82472983, 82372779). Figure 7 was created using Figdraw (www.figdraw.com). We would like to thank the authors who submitted their RNA sequencing data to the GEO database under accession number GSE242541. This study utilized data from the GEO database, which is supported by the NCBI. Author contributions M.S. and H.J. contributed equally to this work. M.S., X.R. and L.Y. designed the research; M.S., H.J. and N.D. performed the research; M.S. wrote the manuscript text; G.L., Y.M. and Y.L. conducted the animal experiments; H.J., Z.H. and Y.Z. made the statistical analysis. All authors read and approved the final manuscript. References Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin. 2024;74(1):12-49. 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Reciprocal interactions between malignant cells and macrophages enhance cancer stemness and M2 polarization in HBV-associated hepatocellular carcinoma. Theranostics. 2024;14(2):892-910. Chen W, Chen M, Hong L, Xiahenazi A, Huang M, Tang N, et al. M2-like tumor-associated macrophage-secreted CCL2 facilitates gallbladder cancer stemness and metastasis. Exp Hematol Oncol. 2024;13(1):83. Zhang R, Shen Y, Zhang Q, Feng X, Liu X, Huo X, et al. TRIM21-mediated Sohlh2 ubiquitination suppresses M2 macrophage polarization and progression of triple-negative breast cancer. Cell Death Dis. 2023;14(12):850. Ji L, Qian W, Gui L, Ji Z, Yin P, Lin GN, et al. Blockade of β-Catenin-Induced CCL28 Suppresses Gastric Cancer Progression via Inhibition of Treg Cell Infiltration. Cancer Res. 2020;80(10):2004-16. Park J, Zhang X, Lee SK, Song N-Y, Son SH, Kim KR, et al. CCL28-induced RARβ expression inhibits oral squamous cell carcinoma bone invasion. J Clin Invest. 2019;129(12):5381-99. Li S, Zhang N, Yang Y, Liu T. Transcriptionally activates CCL28 expression to inhibit M2 polarization of macrophages and prevent immune escape in colorectal cancer cells. Transl Oncol. 2024;40:101842. Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations (Not answered) Supplementary Files Table1.xlsx Table 1 TableS1.xlsx Table S1 TableS2.docx Table S2 TableS3.xlsx Table S3 SupplementaryInformation.docx Supplementary figures SupplementalMaterialWB.tif Original western blots Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: revise 25 Oct, 2025 Review # 3 received at journal 23 Oct, 2025 Review # 2 received at journal 14 Oct, 2025 Reviewer # 3 agreed at journal 10 Oct, 2025 Review # 1 received at journal 04 Oct, 2025 Reviewer # 2 agreed at journal 02 Oct, 2025 Reviewer # 1 agreed at journal 01 Oct, 2025 Reviewers invited by journal 01 Oct, 2025 Submission checks completed at journal 03 Sep, 2025 First submitted to journal 02 Sep, 2025 Editor assigned by journal 02 Sep, 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. 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1","display":"","copyAsset":false,"role":"figure","size":2825858,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eONECUT2 regulates breast cancer stemness via the beta-catenin-dependent pathway.\u003c/strong\u003e \u003cstrong\u003e(A, B) \u003c/strong\u003eThe expression levels of beta-catenin and stemness genes Notch1 and Nanog were verified after transfection with siONECUT2 (A) and an overexpressing plasmid (B) in MDA-231, MCF-7 and BT474 cells using western blotting. \u003cstrong\u003e(C)\u003c/strong\u003e Nuclear beta-catenin, pGSK3β and GSK3β expressions were detected in BC cells following ONECUT2 knockdown by western blotting analysis. \u003cstrong\u003e(D, E) \u003c/strong\u003eMCF-7 cells were transfected with a control siRNA, siONECUT2, or mixed siRNAs (ONECUT2 plus beta-catenin or ONECUT2 plus POU6F2), and collected for FCM to analyze the CD24\u003csup\u003e-\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003e cell population (D) and sphere formation assay (E). \u003cstrong\u003e(F)\u003c/strong\u003e Western blots presenting the expression levels of indicated proteins in BC cells following transfection with specific siRNAs. \u003cstrong\u003e(G)\u003c/strong\u003e Representative mIHC staining images of ONECUT2, POU6F2 and BETA-CATENIN expression in patients with BC (DAPI: blue; ONECUT2: green; POU6F2: red; BETA-CATENIN: yellow). \u003cstrong\u003e(H)\u003c/strong\u003e Scatter plot showing the correlation between ONECUT2 and BETA-CATENIN (n=99, r= -0.2207, p= 0.0281). \u003cstrong\u003e(I) \u003c/strong\u003eKaplan-Meier (KM) survival curves comparing clinical outcomes (DFS, OS) of BC patients between high (red) and low (green) ONECUT2 expression groups. Error bars represent mean ± SD of three independent experiments. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7518380/v1/078018927df18b469a4d1755.jpg"},{"id":93575800,"identity":"1f194f43-8529-428b-8491-7c9af3237f60","added_by":"auto","created_at":"2025-10-15 09:24:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1343190,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eONECUT2 regulates the transcriptional activity of POU6F2. (A, B) \u003c/strong\u003eMDA-231, MCF-7 and BT474 cells were transfected with ONECUT2 siRNAs (A) or its overexpression plasmid (B) for 48h, protein levels of Onecut2 and Pou6f2 were detected by western blotting. \u003cstrong\u003e(C) \u003c/strong\u003eThe correlation analysis of ONECUT2 and POU6F2 expression in BC patients (Left). KM curves showing DFS and OS stratified by POU6F2 expression ( Right; High: red; Low: green). \u003cstrong\u003e(D)\u003c/strong\u003e A Co-IP assay using an anti-ONECUT2 antibody in MDA-231 cells was performed, followed by western blotting to verify the binding among Onecut2, Pou6f2 and beta-catenin. \u003cstrong\u003e(E)\u003c/strong\u003eRelative luciferase activities were determined after co-transfection of MCF-7 cells with luciferase reporter plasmid containing POU6F2 promoter sequences and ONECUT2 overexpression plasmid. \u003cstrong\u003e(F) \u003c/strong\u003eONECUT2-scanned motif logo. Predicted binding sites in the human POU6F2 promoters. Genomics position relative to the transcription start sites of the POU6F2, sequence, and corresponding scores (Left). Binding of ONECUT2 to the POU6F2 promoters in MCF-7 cells was determined by ChIP-PCR (Right). Error bars represent mean ± SD of three independent experiments. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7518380/v1/602fbf8cdedeec40c77649e6.jpg"},{"id":93574346,"identity":"b348e51b-1cc0-474f-99cd-be6d8f05737f","added_by":"auto","created_at":"2025-10-15 09:16:02","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2062070,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePOU6F2 promotes breast cancer stemness via activating beta-catenin.\u003c/strong\u003e \u003cstrong\u003e(A, B) \u003c/strong\u003eWestern blotting was used to assess the protein levels of indicated genes after the BC cells were transfected with POU6F2 siRNAs (A) or an overexpression plasmid (B). \u003cstrong\u003e(C) \u003c/strong\u003eSphere-forming efficiency was evaluated in MCF-7 cells following transfection with siPOU6F2 or a control siRNA. \u003cstrong\u003e(D)\u003c/strong\u003e The proportion of CD24\u003csup\u003e−\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003e cells in MCF-7 cells, with or without POU6F2 knockdown was determined by FCM analysis using anti-CD24-PE and anti-CD44-PerCP-Cy5.5 conjugated antibodies.\u003cstrong\u003e (E) \u003c/strong\u003eFollowing POU6F2 knockdown, MDA-231 cells were treated with DTX or DOXO, and cell viability was measured by CCK-8 assay. Data were normalized to the DMSO control group. \u003cstrong\u003e(F)\u003c/strong\u003e The indicated protein levels were detected in BC cells using western blotting.\u003cstrong\u003e (G)\u003c/strong\u003e The correlation analysis of POU6F2 and BETA-CATENIN expression in BC patients was performed by mHIC analysis (Left). KM curves showed DFS and OS in patients stratified by beta-catenin expression levels (Right, high: red, low: green). \u003cstrong\u003e(H) \u003c/strong\u003eDual-luciferase reporter assays were used to observe the transcriptional regulation of CTNNB1 by POU6F2. \u003cstrong\u003e(I) \u003c/strong\u003ePOU6F2 motif logo and predicted binding sites in the CTNNB1 promoter (Left). ChIP-PCR confirmation of POU6F2 binding to the CTNNB1 promoter in MCF-7 cells (Right). Error bars represent mean ± SD of three independent experiments. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7518380/v1/ff76ff20c817645bec230b62.jpg"},{"id":93572213,"identity":"e6a7436c-3d89-45d7-9975-5a08f6d71527","added_by":"auto","created_at":"2025-10-15 09:08:02","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1964717,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe ONECUT2-POU6F2-beta-catenin axis enhances breast cancer stemness and chemoresistance in vivo.\u003c/strong\u003e \u003cstrong\u003e(A) \u003c/strong\u003eBALB/c tumor models were established by injection of 4T1 cells or 4T1/OC-2 cells into the mammary fat pad. Tumor onset and volume were measured (n=4). \u003cstrong\u003e(B)\u003c/strong\u003e Tumor tissues from BALB/c mouse models were collected for western blotting analyses. \u003cstrong\u003e(C)\u003c/strong\u003eXenograft tumors generated in NSG mice by injecting MDA-231 and MDA-231/OC-2 cells. Tumor volume was measured, with DTX treatments marked by arrows (Left). The protein level of ONECUT2 in NSG tumor models was measured (Right). \u003cstrong\u003e(D, E) \u003c/strong\u003eWestern blots of indicated proteins in tumor tissues from (D) MDA-231 mice (n=4) and (E) MDA-231/OC-2 mice (n=3) collected before and after DTX treatment. \u003cstrong\u003e(F)\u003c/strong\u003e The expression levels of ONECUT2, POU6F2 and beta-catenin in the MDA-231 mice tissues pre- and post-DTX were detected by IHC staining. Error bars represent mean ± SD of three independent experiments. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7518380/v1/a0ec8c06219d74527030518d.jpg"},{"id":93572214,"identity":"6b0fa752-f1dd-4a33-ab2f-d65a44bddfdf","added_by":"auto","created_at":"2025-10-15 09:08:02","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1861114,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eONECUT2 drives macrophage polarization to M2 phenotype in the breast cancer microenvironment.\u003c/strong\u003e \u003cstrong\u003e(A-C) \u003c/strong\u003eFCM was used to assess dynamic changes in immunocyte subsets (M2-type macrophages, macrophages, CD3\u003csup\u003e+\u003c/sup\u003eT cells, CD4\u003csup\u003e+\u003c/sup\u003eT cells, CD8\u003csup\u003e+\u003c/sup\u003eT cells, B cells, NK cells and Tregs) within the tumor tissues (A, B) and spleens (C) of 4T1/OC2 and 4T1 mice. \u003cstrong\u003e(D)\u003c/strong\u003e THP-1 cells were co-cultured with MDA-231-conditional supernatant, and the CD163\u003csup\u003e+\u003c/sup\u003eCD68\u003csup\u003e+\u003c/sup\u003e population was assessed by FCM analysis. \u003cstrong\u003e(E, F)\u003c/strong\u003e RT-qPCR analysis for the mRNA levels of CD163 and CD206 (E), as well as IL-10 and Arg-1 (F) in MDA-231 cells with ONECUT2 inhibition or overexpression, compared to their respective control groups. Error bars represent mean ± SD of three independent experiments, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7518380/v1/cadf6f5c52f74d7090d1ea89.jpg"},{"id":93572220,"identity":"c2489c65-e391-498c-a4c7-7351fae18998","added_by":"auto","created_at":"2025-10-15 09:08:02","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2534935,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCL28 is a direct target of ONECUT2 required for M2 macrophage polarization. (A) \u003c/strong\u003eThe publicly available RNA-seq data (GSE242541) was analyzed to identify altered genes following ONECUT2 overexpression in MCF-7 and BT474 cells. \u003cstrong\u003e(B)\u003c/strong\u003e RT-qPCR analysis for CCL28 mRNA levels with ONECUT2 overexpression (upper panel) or knockdown (lower panel) in BC cell lines. \u003cstrong\u003e(C, D) \u003c/strong\u003eWestern blotting was used to detect the expression of CCL28 after overexpressing (C) or silencing ONECUT2 (D). \u003cstrong\u003e(E) \u003c/strong\u003eCCL28 expression was evaluated by western blotting in the tumor tissues of BALB/c mouse models. \u003cstrong\u003e(F)\u003c/strong\u003e IHC analyses were performed to quantify CCL28 expression in MDA-231 or MDA-231/OC-2 xenograft tumors. \u003cstrong\u003e(G)\u003c/strong\u003e Western blotting analysis of CCL28 level in MDA-231 cells transfected with indicated siRNAs. \u003cstrong\u003e(H) \u003c/strong\u003eFCM analysis were used to explore the CD163\u003csup\u003e+\u003c/sup\u003eCD68\u003csup\u003e+\u003c/sup\u003e population in treated MDA-231 cells. \u003cstrong\u003e(I)\u003c/strong\u003e Dual-luciferase reporter assay for detecting the activity of CCL28 promoters in MCF-7 cells which were transfected with ONECUT2 overexpression or control vector. \u003cstrong\u003e(J)\u003c/strong\u003e ONECUT2 motif logo and predicted binding sites in the CCL28 promoter (Left). ChIP-PCR confirmation of ONECUT2 binding to the CCL28 promoter in MCF-7 cells (Right). \u003cstrong\u003e(K)\u003c/strong\u003e RT-qPCR and \u003cstrong\u003e(L)\u003c/strong\u003e western blotting of CCR3 and CCR10 in MDA-231 cells with ONECUT2 knockdown or overexpression. Error bars represent mean ± SD of three independent experiments *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7518380/v1/9a389239ef8e23b5b229d124.jpg"},{"id":93572217,"identity":"d4ff230c-36fe-42f4-adb8-411648c874ec","added_by":"auto","created_at":"2025-10-15 09:08:02","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":782968,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram. \u003c/strong\u003eONECUT2 regulates breast cancer stemness characteristics and chemoresistance via the POU6F2-beta-catenin axis and facilitates macrophage polarization to M2 phenotype through the CCL28-CCR10 pathway.\u003c/p\u003e","description":"","filename":"Figure7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7518380/v1/65bb495ee7a04a664f715c33.jpg"},{"id":93576349,"identity":"a5708f9e-a154-4f3c-a2a0-8775b6ebc183","added_by":"auto","created_at":"2025-10-15 09:32:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14434538,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7518380/v1/714feb9f-8a1f-4a3d-afd0-3956da5b66d7.pdf"},{"id":93572206,"identity":"2ce02ebc-d716-4b01-b478-b08c80d56d27","added_by":"auto","created_at":"2025-10-15 09:08:02","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11883,"visible":true,"origin":"","legend":"\u003cp\u003eTable 1\u003c/p\u003e","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7518380/v1/a023d4a924867b80b2e5da2a.xlsx"},{"id":93574347,"identity":"79536319-4845-4829-813e-ecb2c207bda6","added_by":"auto","created_at":"2025-10-15 09:16:02","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12207,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1\u003c/p\u003e","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7518380/v1/a0f002f03afabcce0c6c7c69.xlsx"},{"id":93572209,"identity":"c84e8c78-8f81-4bf0-9c36-290559df47df","added_by":"auto","created_at":"2025-10-15 09:08:02","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":29202,"visible":true,"origin":"","legend":"Table S2","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7518380/v1/732e416f3a344ba979dc6038.docx"},{"id":93572216,"identity":"075fc926-7adc-464a-85d4-d8e6e9be2da7","added_by":"auto","created_at":"2025-10-15 09:08:02","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":402701,"visible":true,"origin":"","legend":"Table S3","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7518380/v1/008f79db83cc2e19a5249fb5.xlsx"},{"id":93575801,"identity":"335ab200-8c98-4809-9b26-795de944c8ca","added_by":"auto","created_at":"2025-10-15 09:24:03","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1205665,"visible":true,"origin":"","legend":"Supplementary figures","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7518380/v1/83f3c650439b3795864947d9.docx"},{"id":93572230,"identity":"1c2d5bc9-2af2-49c4-9f2e-1fde79597701","added_by":"auto","created_at":"2025-10-15 09:08:03","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":51126042,"visible":true,"origin":"","legend":"Original western blots","description":"","filename":"SupplementalMaterialWB.tif","url":"https://assets-eu.researchsquare.com/files/rs-7518380/v1/c8c999cfdb9c0f899aabbde3.tif"}],"financialInterests":"(Not answered)","formattedTitle":"ONECUT2 governs the POU6F2-beta-catenin axis to modulate cancer stemness and drives the CCL28-dependent pathway for macrophage polarization in breast cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer (BC) is the predominant malignant tumor among women and has a profound impact on their health [1]. Cancer stem cells (CSC) exhibit intrinsic resistance to chemotherapeutic agents, which contributes to tumor progression and recurrence in BC. Exploring how chemotherapy regulates BC stemness is of great significance for reversing chemoresistance and ultimately improving patient prognosis. In our previous study [2] , we identified a novel mechanism by which chemotherapy induces BC cells to secrete extracellular vesicles (EVs) that serve as critical mediators of therapeutic resistance. These EVs targeted the transcription factor One Cut Homeobox 2 (ONECUT2), thereby inducing a CSC-like phenotype and chemoresistance in BC. ONECUT2, a core member of the ONECUT family, is a transcription factor that is involved in multidirectional differentiation [3]. ONECUT2 plays a role in various oncogenic processes, including tumor proliferation, metastasis, epithelial-mesenchymal transition (EMT), angiogenic signaling, resistance to endocrine therapy, and subtype switching in BC [4-6]. However, the effects of ONECUT2 on CSC properties are complex and involve interactions with multiple signaling pathways and molecular mechanisms that collectively reshape CSC phenotypes. As a transcription factor, ONECUT2 modulates CSC traits by regulating the downstream genes PPP2R4 [7] or TFPI [8] in gastric cancer (GC). Another study [4] elucidates the roles of ONECUT2 in stemness maintenance, verifying beta-catenin as a crucial downstream effector. In non-small cell lung cancer (NSCLC) [9], ONECUT2 has emerged as a vital downstream effector in CSC driven by lncRNA THUMPD3-AS1, highlighting its potential as a therapeutic target in NSCLC. Although our findings suggest a regulatory role of ONECUT2 in CSC properties, the underlying molecular mechanisms are poorly understood. This knowledge gap underscores the need to fully elucidate the mechanism by which ONECUT2 mediates functional plasticity in CSC, thereby paving the way for the development of novel therapeutic strategies. In our study, we found that ONECUT2 transcriptionally targets POU6F2 (POU domain, class 6, transcription factor 2), thereby triggering beta-catenin and modulating cancer stemness and chemoresistance in BC cell lines, animal models, and clinical specimens.\u003c/p\u003e\n\u003cp\u003eEmerging evidence indicates that the interplay between CSC and the tumor immune microenvironment (TIME) drives tumor progression and immune evasion [10-13] . In NSCLC, ONECUT2 serves as a master mediator of the exhausted CD8\u003csup\u003e+\u003c/sup\u003eT cell phenotype [14]; thus, it has been identified as a critical regulator of cytotoxic immune responses and a novel therapeutic target for checkpoint inhibition. Another study identified ONECUT2 as a direct target of T-bet [15], establishing a central role in the differentiation of T-helper (Th) progenitor cells to Th1/Th2 effector cells. However, the precise role of ONECUT2 in the BC TIME remains unknown. Here, we observed through in vivo and in vitro experiments that ONECUT2 functions as a key regulator of M2-type macrophage polarization, which reprograms the TIME. This study further elucidates the regulatory network of the immunosuppressive microenvironment and screens for potential therapeutic targets to enhance the efficacy of immunotherapy in BC.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eCells and constructs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human BC cell lines (MDA-231, MCF-7 and BT474), mouse BC cell line (4T1) and human THP-1 cell line were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in the recommended media. All cancer cell lines have been authenticated by STR genotyping and proved to be free of mycoplasma contamination. MDA-231 cells with stable ONECUT2 overexpression (MDA-231/OC-2) were generated by ONECUT2 overexpression plasmid (OriGene; Rockville, MD), and subsequently selected by G418 solution. 4T1 cells with stable ONECUT2 overexpression (4T1/OC-2) were generated by transduction with lentivirus carrying pLVX-Onecut2 (mouse, NM_194268.3), followed by puromycin selection, and control cells were generated using pLVX empty vector. The siRNAs targeting ONECUT2, POU6F2, and CTNNB1, along with negative control siRNA, were purchased from Qiagen (Venlo, Netherlands). RNA or DNA was transfected using Lipofectamine RNAiMAX (Invitrogen; Carlsbad, CA) or Lipofectamine 3000 (Invitrogen; Carlsbad, CA) following the manufacturer’s protocols.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatients and tissue samples \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue microarrays (TMAs) comprising 99 BC samples coupled with patients’ survival data were acquired from Shanghai Outdo Biotech (Shanghai, China). All experimental procedures were conducted following approval by the Ethics Committee of Tianjin Medical University Cancer Institute and Hospital (E2021075) and conducted in accordance with the Declaration of Helsinki. The clinical parameters of the patients were shown in Table S1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReal-time quantitative PCR\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and Western blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA extraction by TRIZOL (Invitrogen; Carlsbad, CA) and real-time quantitative PCR (RT-qPCR) were performed as previously described [2], with β-ACTIN as a control (primer sequences in Table S2). Proteins were extracted using RIPA lysis buffer, with antibodies listed in Table S2. Nuclear proteins were extracted using the Nuclear Protein Extraction Kit (Bestbio; Shanghai, China) following the manufacturer’s protocol.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSphere formation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTreated MCF-7 cells were harvested and re-suspended in serum-free medium. The single-cell suspension was diluted to a density of 2 000 cells per well and seeded into ultralow attachment 6-well plates. The number of spheres was counted on day 14, and the sphere-forming efficiency was calculated using a diameter ≥70μm as the criterion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme-linked immunosorbent assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCultured supernatants were collected and assayed for enzyme-linked immunosorbent assay (ELISA) by ElaBoXTMHuman MEC (CCL28) ELISA Kit (Solarbio; Beijing, China) following the manufacturer’s instructions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSingle-cell suspensions were prepared from tumor tissues/spleens of mice or treated BC cells, followed by staining with antibodies (listed in Table S2) for flow cytometry (FCM) analyses. All samples were analyzed using a FACS with 3 replicates, and at least 10 000 events were acquired for each sample.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDual-luciferase reporter assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMCF-7 cells were seeded at about 60% confluence in 24-well plates. For promoter reporters, 0.5μg pGL3-Basic luciferase reporters were transfected into MCF-7 cells with ONECUT2 or POU6F2 overexpression. Empty pCMV6-Entry or pcDNA3.1(+) vector were used as control groups. The luciferase activities were determined using the Dual-Luciferase Reporter Assay System (Promega), as described previously [2]. Primer sequences are listed in Table S2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChIP-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMCF-7 cells were harvested and performed using the ChIP Assay kit (Cell Signaling Technology, Massachusetts, USA.) under the manufacturer’s instructions. The DNA enrichment was assessed by real-time PCR using the specific primers (detailed in Table S2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-Immunoprecipitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe co-immunoprecipitation (Co-IP) assay was performed using the Epizyme Co-IP Kit (YJ201; Epizyme Biotech, China) according to the manufacturer’s instructions. In brief, lysates of 1×10\u003csup\u003e7\u003c/sup\u003e MDA-231 cells were incubated with anti-ONECUT2 antibody or IgG isotype control overnight at 4°C, subsequently co-incubated with protein A/G magnetic beads for 6 h, and further eluted for western blotting. Table S2 displays the used antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell viability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell viability was assessed using Cell Counting Kit-8 (CCK8, MedChemExpress, China). Briefly, MDA-231 cells were seeded at 2 500 cells/well using flat-bottomed 96-well culture plates and treated with docetaxel (DTX, 4 nM, Beyotime Biotechnology, China) or doxorubicin (DOXO, 125 nM, Sigma-Aldrich, USA). At indicated time points, 10μl CCK8 solution was added to each well followed by 2 h incubation at 37℃, and absorbance at 450 nm was measured.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry (IHC) and multiplex IHC staining (mIHC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFormaldehyde-fixed, paraffin-embedded mouse tumor tissues was processed for IHC as previously described [16]. The antibodies were listed in Table S2. The stained slides were evaluated according to two criteria: 1) staining intensity (-: 0, +: 1, ++: 2, +++: 3) and 2) percentage of staining-positive tumor cells (0%: 0, 1~29%: 1, 30~69%: 2, ≥ 70%: 3). The intensity and percentage scores were multiplied to generate a final score, which were used in statistical analyses. mIHC staining was conducted by PANO 7-plex IHC Kit (Panovue, Beijing, China). DAPI was used for nuclear localization. The antibodies and their dilution ratios were listed in Table S2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Institutional Animal Care and Use Committee of Tianjin Medical University Cancer Institute and Hospital (E2021128). Six-week-old female NOD/SCID/IL2Rγ-null (NSG) mice and BALB/c mice were obtained from the Weitonglihua Corporation (Beijing, China). For the 4T1 mouse models, 4T1/OC-2 or 4T1 cells were injected into the mammary fat pad, and the tumor volume was measured using calipers. Tumors were harvested 20 days after injection. For the NSG mouse models, MDA-231/OC-2 or MDA-231 cells were injected into the mammary fat pads. Once the tumor volume reached ~300 mm\u003csup\u003e3\u003c/sup\u003e,\u0026nbsp;the mice were divided into two groups: one group was sacrificed immediately\u0026nbsp;(pre-DTX group),\u0026nbsp;and the other group\u0026nbsp;(post-DTX group) was treated\u0026nbsp;with DTX\u0026nbsp;(15 mg/kg) for three weeks and sacrificed three days after\u0026nbsp;the\u0026nbsp;final DTX treatment. Tumor tissues were cut into two pieces for subsequent experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGraphPad Prism version 10.4.2 (GraphPad Software, USA) was used for graph drawing and statistical analysis. The results were derived from at least three independent experiments. Statistical significance between groups was assessed using Student's unpaired t-test, Student's paired t-test, Mann-Whitney test, one-way analysis of variance (ANOVA) for multiple comparisons, Pearson's correlation analysis, χ² test, log-rank test, over-representation analysis, and the Cox proportional hazards regression models, as appropriate. Statistical significance was set at p\u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eONECUT2 regulates breast cancer stemness via the beta-catenin-dependent pathway\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur previous study showed that ONECUT2 serves as a crucial determinant of CSC [2]; however, the underlying molecular mechanism remains poorly understood. Since the beta-catenin signaling pathway acts as a core set, with its target genes commonly serving as well-known stem-cell markers [17], we examined whether ONECUT2 could affect beta-catenin expression and clarify the regulatory mechanism. In MDA-231, MCF-7, and BT474 cell lines, ONECUT2 inhibition activated beta-catenin and upregulated stemness genes Notch1 and Nanog (Fig.1A). Conversely, ONECUT2 overexpression exhibited the opposite effect (Fig. 1B). We further observed a significant accumulation in nuclear beta-catenin rather than pGSK3β in BC cells (Fig. 1C), indicating that ONECUT2 activates the transcriptional activity of beta-catenin. However, this regulation did not directly bind to the promoter of beta-catenin, as evidenced by a luciferase assay showing that ONECUT2 had no effect on the CTNNB1 (encoding beta-catenin)\u0026nbsp;promoter (Fig. S1).\u0026nbsp;Beta-catenin attenuated the effect of ONECUT2 on the CD24\u003csup\u003e-\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003e population known to be enriched in CSC (Fig.1D), sphere-forming efficiency (Fig. 1E, S2), and the expression of stemness gene Notch1 and Nanog (Fig. 1F) in BC cells. mIHC staining confirmed a significant inverse correlation (r=-0.2207, p=0.0281) between the expression of ONECUT2 and beta-catenin in BC patients (Fig. 1G, H). Disease-free survival (DFS) and overall survival (OS) were significantly shorter in the ONECUT2-low expression cohort (DFS, p\u0026lt;\u0026nbsp;0.0001; OS, p= 0.0455;\u0026nbsp;Fig. I, Table 1). Collectively, these findings suggest that ONECUT2 exerts its CSC-modulatory role via a beta-catenin-dependent pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eONECUT2 regulates the transcriptional activity of POU6F2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe potential target genes of ONECUT2 were predicted using the Gene Transcription Regulation Database (GTRD) (http://gtrd.biouml.org/). When setting the transcription factor-binding site at the promoter [-2600, +100], we observed that POU6F2 was a promising target gene (Table S3). Given that POU6F2, reported to be regulated by ONECUT2 in embryonic retinas [3], was upregulated in ONECUT2-knockdown BC cells in our previous study\u0026nbsp;[2], we conducted experiments to observe whether POU6F2 serves as a direct transcriptional target of ONECUT2, thereby mediating the regulatory effects of ONECUT2 on beta-catenin. In BC cells,\u0026nbsp;ONECUT2 knockdown\u0026nbsp;promoted POU6F2 expression (Fig. 2A), whereas ONECUT2 overexpression reduced POU6F2 expression (Fig. 2B).\u0026nbsp;mIHC staining showed that ONECUT2 and POU6F2 exhibited a significant negative correlation (r=-0.2021, p=0.0449). DFS (p=0.0003) and OS (p=0.0031) were significantly prolonged in patients with low POU6F2 levels (Fig. 2C, Table 1). Further, we employed a\u0026nbsp;Co-IP assay to confirm the binding among Onecut2, Pou6f2, and beta-catenin (Fig. 2D). Dual-luciferase reporter assays (Fig. 2E) and ChIP-PCR analysis (Fig. 2F) demonstrated that ONECUT2 regulates POU6F2 transcriptional activity and directly binds to its promoter region. These results provide conclusive evidence that POU6F2 is a direct transcriptional target of ONECUT2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePOU6F2 promotes breast cancer stemness via activating beta-catenin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on these findings, we speculated that POU6F2 may play a core role in exerting ONECUT2-mediated beta-catenin activation and CSC promotion in BC. POU6F2 deficiency in MDA-231, MCF-7, and BT474 cells resulted in decreased protein levels of beta-catenin and stemness-associated genes, including Notch1 and Nanog (Fig. 3A). Conversely, POU6F2 overexpression markedly increased the expression of these proteins (Fig. 3B),\u0026nbsp;suggesting a regulatory function of POU6F2 on beta-catenin and the maintenance of CSC. Furthermore, the sphere formation ability (Fig. 3C) and the CD24\u003csup\u003e-\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003e cell population (Fig. 3D) decreased with POU6F2 knockdown in MCF-7 cells.\u0026nbsp;Following POU6F2 inhibition, we observed reduced resistance to DTX and DOXO in MDA-231 cells (Fig. 3E), underscoring POU6F2’s critical roles in promoting chemoresistance in BC\u0026nbsp;cells. Furthermore, we observed a notable reduction of beta-catenin and stemness genes (Fig. 3F), the CD24\u003csup\u003e-\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003e cell population (Fig. 1D), and the sphere formation ability (Fig. 1E) in the ONECUT2-deficient group with POU6F2 knockdown. As shown in Fig. 3G, POU6F2 demonstrated a strong positive correlation with beta-catenin (r=0.4496, p＜0.0001). Furthermore, patients with elevated beta-catenin expression exhibited significantly shortened DFS (p=0.0074), while no statistical association was observed between beta-catenin levels and OS (p=0.4479, Table 1). Mechanistically, using dual-luciferase reporter assays, we\u0026nbsp;found that POU6F2 served\u0026nbsp;as a transcriptional activator of CTNNB1, revealing significantly increased promoter activity upon POU6F2 overexpression (Fig. 3H).\u0026nbsp;\u0026nbsp;ChIP-PCR analysis further confirmed\u0026nbsp;that\u0026nbsp;POU6F2 binds to the promoter region of CTNNB1 (Fig. 3I). Taken together, these data highlight POU6F2 as a crucial mediator in activating beta-catenin, thereby promoting cancer stemness and chemoresistance in BC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe ONECUT2-POU6F2-beta-catenin axis enhances breast cancer stemness and chemoresistance in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further evaluate the\u0026nbsp;role of the ONECUT2-POU6F2-beta-catenin axis in modulating BC stemness and chemotherapy resistance, we constructed BALB/c mouse models bearing 4T1/OC-2 cells and 4T1 cells (Fig. 4A). A significant decrease of Pou6f2 and beta-catenin expression was observed in 4T1/OC-2 tumors relative to 4T1 tumors (Fig. 4B). Another NSG mouse model was generated, comprising MDA-231 cells and MDA-231/OC-2 cells (Fig. 4C), to analyze the role of the ONECUT2-POU6F2-beta-catenin axis in mediating CSC and chemoresistance in vivo. In MDA-231 tumor models, DTX treatment resulted in decreased Onecut2 expression, accompanied by increased expression of Pou6f2, beta-catenin, and Nanog (Fig. 4D, 4F), whereas it had no effect in MDA-231/OC-2 tumors (Fig. 4E). These results further support the critical role of the ONECUT2-POU6F2-beta-catenin axis in maintaining cancer stemness and chemoresistance in BC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eONECUT2 drives macrophage polarization to M2 phenotype in the breast cancer microenvironment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGrowing evidence suggests that there are complex interactions between CSCs and TIME [18, 19]. To determine the regulatory roles of ONECUT2 in the TIME, FCM was used to assess dynamic changes in immunocyte subsets within the tumor tissues and spleens\u0026nbsp;of 4T1/OC-2 and 4T1 mice. We observed a notable reduction in\u0026nbsp;M2 macrophage infiltration and an increase in the proportion of B cells in 4T1/OC-2 tumors (Fig. 5A, B). Spleen tissues\u0026nbsp;from 4T1/OC-2 mice exhibited\u0026nbsp;significant reductions in M2 macrophages and CD4\u003csup\u003e+\u003c/sup\u003eT cell infiltration, accompanied by elevated percentages of CD3\u003csup\u003e+\u003c/sup\u003eT cells, CD8\u003csup\u003e+\u003c/sup\u003eT cells, B cells, and regulatory T cells (Tregs) (Fig. 5C, S3). Consistent results were obtained for THP-1-derived macrophages. ONECUT2 inhibition increased the CD163\u003csup\u003e+\u003c/sup\u003e M2 macrophage population (Fig. 5D left, S4 upper\u0026nbsp;panel) and the expression of\u0026nbsp;the\u0026nbsp;pro-cancer M2 markers CD163 and CD206 (Fig. 5E,\u0026nbsp;left). Conversely, ONECUT2 overexpression demonstrated opposite effects (Fig. 5D right, S4 lower panel, and 5E right). As shown in Fig. 5F, silencing ONECUT2 increased the mRNA levels of IL-10 and Arg-1 produced by macrophages, whereas ONECUT2 overexpression\u0026nbsp;was correlated with reduced mRNA levels of these genes. Our study indicated that ONECUT2 acts as a vital regulator of M2-type macrophage polarization in BC, underscoring its potential as a therapeutic target to reverse the immunosuppressive microenvironment and enhance\u0026nbsp;the efficacy of immunotherapy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCCL28 is a direct target of ONECUT2 required for M2 macrophage polarization\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the regulatory mechanism of ONECUT2 in M2 macrophage polarization, we analyzed the publicly available RNA-seq data from Irene et al. (GEO accession: GSE242541) [6] and observed that CCL28 was downregulated in MCF-7 and BT474 cells overexpressing ONECUT2 (Fig. 6A). Given that CCL28 [20] participates in BC progression by modulating proliferation, invasion, and metastasis, we investigated how ONECUT2 regulates CCL28 expression and subsequently reprograms macrophages toward the M2 phenotype. Decreased CCL28 expression in response to ONECUT2 overexpression was confirmed in BC cell lines at both the RNA (Fig. 6B, upper panel) and protein levels (Fig. 6C), whereas ONECUT2 deficiency produced the opposite effects (Fig. 6B lower panel, 6D). ELISA confirmed that ONECUT2 knockdown significantly elevated the secretion of CCL28 (Fig. S5). Furthermore, CCL28 expression was downregulated in both 4T1/OC-2 tumors (Fig. 6E) and MDA-231/OC-2 xenografts (Fig. 6F) compared with their respective controls (4T1 and MDA-231). Western blotting analysis showed that CCL28 overexpression partially rescued the effect of ONECUT2 on CCL28 expression in MDA-231 cells (Fig. 6G). Increased CCL28 expression led to an increase in the CD163\u003csup\u003e+\u003c/sup\u003e M2 macrophage population and elevated expression of M2 markers (CD163 and CD206), which were partially blocked by ONECUT2 overexpression (Fig. 6H, S6A). Based on predictions from the JASPAR database, dual-luciferase reporter assays demonstrated that ONECUT2 significantly repressed the transcriptional activity of CCL28 in MCF-7 cells (Fig. 6I). ChIP-PCR further confirmed that ONECUT2 binds to the CCL28 promoter (Fig. 6J). Additionally, to investigate the receptors of CCL28 involved in the regulation of M2-type macrophage polarization, we examined the expression of CCR3 and CCR10 (known as CCL28 receptors). Knockdown of ONECUT2 in MDA-231 cells increased the RNA and protein levels of CCR10 in macrophages, whereas ONECUT2 overexpression induced the opposite effect. However, there were no significant changes in the CCR3 protein levels (Fig. 6K, 6L). The induction of CCR10 by CCL28 overexpression was reversed by ONECUT2 overexpression in MDA-231 cells (Fig. S6B). These findings suggest that CCR10 might act as a key molecule in ONECUT2-CCL28-mediated regulation of macrophage polarization to the M2 phenotype. Collectively, these results indicate that ONECUT2 transcriptionally targets CCL28, thereby driving macrophage polarization to an immunosuppressive M2-like phenotype. The ONECUT2-CCL28-CCR10 signaling axis reprograms macrophage polarization and modulates the TIME in BC.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, we demonstrate a specific mechanism by which ONECUT2 regulates cancer stemness characteristics and affects the immunosuppressive environment in BC. Our current study reveals novel mechanisms underlying cancer cell self-renewal capacity and immunoregulatory function, which alleviate chemoresistance and improve survival outcomes in patients with BC. Growing evidence suggests that ONECUT2 is a multifaceted regulator of CSC properties and that its expression is significantly correlated with clinical prognosis\u0026nbsp;[7, 21-23]. As shown in our previous study, BC-derived\u0026nbsp;EVs\u0026nbsp;promote CSC traits and chemotherapy resistance\u0026nbsp;by downregulating ONECUT2\u0026nbsp;expression. Other studies\u0026nbsp;[4, 9]\u0026nbsp;further reveal that ONECUT2 is an effective regulator in maintaining cancer stemness\u0026nbsp;and chemoresistance, while the precise mechanism remains to be fully elucidated. Recent studies have shown that ONECUT2 is a master regulator of CSCs properties through transcriptional reprogramming. ONECUT2 activates the transcriptional activity of TFPI in GC\u0026nbsp;[8], thereby contributing to CSC traits and oxaliplatin resistance. Another study demonstrates that ONECUT2 inhibits PPP2R4 transcription\u0026nbsp;[7], which in turn increases AKT/beta-catenin phosphorylation, thereby inducing GC stemness. Furthermore, ONECUT2 exerts a positive association with Wnt/beta-catenin signaling pathway activation\u0026nbsp;[21], revealing a significant correlation between ONECUT2 expression and beta-catenin pathway activity. The beta-catenin signaling was reported as a core pathway in maintaining CSC phenotype, with its target genes commonly serving as established stem-cell markers\u0026nbsp;[24]. Modulation of homeostasis facilitates stem-cell self-renewal balance\u0026nbsp;[25]. In our current study, we identified beta-catenin functions as a central effector in ONECUT2-driven CSC maintenance and chemoresistance. ONECUT2 inhibition activated beta-catenin and induced a CSC phenotype in BC, suggesting beta-catenin as a pivotal target in ONECUT2-mediated cancer stemness property.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePOU6F2 was originally cloned from the human retina and is known as retina-derived POU-domain factor-1(RPF-1). The current understanding of POU6F2 is largely confined to the differentiation and morphogenesis of the kidney [26] and eye cornea [27]. Although several studies have elucidated the function of POU6F2 in tumor biology, its regulatory role in maintaining cancer stemness remains largely unexplored. POU6F2 exhibits dual regulatory roles in cancer, with tumor-promoting or tumor-suppressive functions influenced by microenvironmental factors across malignancies. In gastric adenocarcinoma with liver metastasis [28] and renal cell cancer [29], it functions as an oncogene, and upregulated expression is associated with a worse clinical prognosis. Conversely, POU6F2 exerts tumor-suppressive effects in Wilms’ tumor [30] and prolactinoma [31], which serve as potential targets for cancer treatment. In this study, we first demonstrated that ONECUT2 inhibition activates the transcriptional activity of POU6F2, subsequently inducing CSC phenotype and chemotherapy resistance through a beta-catenin-dependent pathway. These findings add a mechanism of mediating chemotherapy-driven BC stemness through the ONECUT2-POU6F2-beta-catenin axis, and suggest novel therapeutic targets for reversing chemoresistance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe immunosuppressive TIME is a major barrier to effective cancer immunotherapies. Reprogramming tumor-associated macrophages (TAMs) into an antitumor phenotype is a promising strategy. Studies [32, 33] have revealed that TAMs engage in functional crosstalk with the CSC phenotype. TAMs undergo preferential polarization toward the M2 phenotype, which acquires potent immunosuppressive activity that mediates pro-tumorigenic functions in BC [34]. In our study, we analyzed the changes in various immunocyte subsets in 4T1/OC-2 BALB/c mouse models and found a significant reduction in macrophage polarization towards the M2 type. Functionally, as evidenced by the RNA-Sequencing results shown in Fig. 6A, we figured out that CCL28 is a key mediator of ONECUT2-driven M2-type macrophage polarization. ONECUT2 knockdown leads to the transcriptional activation of CCL28, which subsequently induces macrophage polarization via the CCL28-CCR10 signaling axis, thereby creating an immunosuppressive TIME in BC. CCL28, a member of the CC chemokine subfamily, has been implicated in the promotion of tumor progression through immune regulation and EMT modulation [35, 36] . While a previous study [37] on colorectal cancer reported that CCL28 suppressed macrophage polarization to the M2 phenotype and inhibited tumor progression, our findings revealed a paradoxical pro-tumorigenic role in BC, where CCL28 upregulation enhanced M2-type polarization through a CCR10-dependent mechanism. This discrepancy may stem from the heterogeneity of the TIME and the presence of other mechanisms and factors that influence the interplay between CCL28-M2 macrophage, which requires further exploration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur findings are the first to demonstrate the specific characteristics of ONECUT2 that shape transcriptional profiles linked to CSC traits and TIME in BC (Fig. 7). Conceivably, targeting pivotal molecules in the ONECUT2-POU6F2-beta-catenin and ONECUT2-CCL28-CCR10 signaling pathways could reduce cancer cell stemness as well as enhance the efficacy of chemotherapy and immunotherapy in BC.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated in this study are available upon request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (No. 82103001, 82472983, 82372779). Figure 7 was created using Figdraw (www.figdraw.com). We would like to thank the authors who submitted their RNA sequencing data to the GEO database under accession number GSE242541. This study utilized data from the GEO database, which is supported by the NCBI.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.S. and H.J. contributed equally to this work. M.S., X.R. and L.Y. designed the research; M.S., H.J. and N.D. performed the research; M.S. wrote the manuscript text; G.L., Y.M. and Y.L. conducted the animal experiments; H.J., Z.H. and Y.Z. made the statistical analysis. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSiegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin. 2024;74(1):12-49.\u003c/li\u003e\n\u003cli\u003eShen M, Dong C, Ruan X, Yan W, Cao M, Pizzo D, et al. 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Transl Oncol. 2024;40:101842.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7518380/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7518380/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Cancer stem cells (CSC) and the immunosuppressive microenvironments are key drivers of breast cancer (BC) progression and drug resistance. However, the molecular mechanisms by which One Cut Homeobox 2 (ONECUT2) governs CSC and the tumor immune microenvironment (TIME) remains largely unknown. Given the critical knowledge gap, we sought to investigate ONECUT2’s regulatory impact on CSC properties and TIME profiles using breast cancer cell lines, animal models, and clinical specimens. Here, we demonstrated that ONECUT2, a core transcription factor, mediates CSC characteristics and reprograms the TIME to drive macrophage polarization to the M2-type, a tumor-promoting state. Mechanistically, ONECUT2 inhibition transcriptionally activated POU6F2, which subsequently triggered beta-catenin, thereby enhancing CSC properties and chemoresistance in BC. With respect to modulating the immune microenvironment, ONECUT2 can govern macrophage polarization, identifying CCL28 as a transcriptional target of ONECUT2 required for M2-type macrophage polarization, and CCR10 as a key receptor involved in immune modulation. These findings highlight the critical involvement of ONECUT2 in modulating BC stemness via targeting the POU6F2-beta-catenin axis and managing macrophage polarization to M2 phenotype through the CCL28-CCR10 pathway. Our study suggests that ONECUT2 modulates cancer stemness and the immune microenvironment, and that targeting it along its downstream axis may provide an effective approach for BC treatment.","manuscriptTitle":"ONECUT2 governs the POU6F2-beta-catenin axis to modulate cancer stemness and drives the CCL28-dependent pathway for macrophage polarization in breast cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 09:07:57","doi":"10.21203/rs.3.rs-7518380/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-10-25T11:39:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-10-24T03:36:15+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-10-14T14:20:46+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-10T12:36:58+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-10-05T02:19:24+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-02T08:43:29+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-01T23:34:56+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-10-01T18:08:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-03T09:55:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2025-09-02T13:46:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-02T13:46:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8fddf230-c524-4dce-b9e3-f871fa9f0f1d","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":55647229,"name":"Health sciences/Diseases/Cancer/Breast cancer"},{"id":55647230,"name":"Health sciences/Diseases/Cancer/Cancer microenvironment"}],"tags":[],"updatedAt":"2026-04-21T04:36:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-15 09:07:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7518380","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7518380","identity":"rs-7518380","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}