RBBP4 orchestrates glycolytic reprogramming and NF-κB-mediated immune evasion in triple-negative breast cancer

RBBP4 orchestrates glycolytic reprogramming and NF-κB-mediated immune evasion in triple-negative 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 Research Article RBBP4 orchestrates glycolytic reprogramming and NF-κB-mediated immune evasion in triple-negative breast cancer Xuyuan Dong, Hongyan Xu, Pengcheng zou, Jianun Lei, Shan Shao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7280626/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective: To investigate the role of retinoblastoma binding protein 4 (RBBP4) in triple-negative breast cancer (TNBC), an aggressive tumor lacking targeted treatments, and explore its potential as a therapeutic target. Methods: The study analyzed RBBP4 expression in TNBC tumors and its association with patient survival. Experimental approaches included assessing the impact of RBBP4 on in vitro cellular proliferation and in vivo tumor growth, as well as invasion and migration. Transcriptomic analyses were performed to examine RBBP4-driven metabolic reprogramming. Molecular interactions between RBBP4 and RelB, and the effects of glycolysis-derived lactate on epigenetic regulation, were also investigated. Results: RBBP4 was upregulated in TNBC tumors, with higher levels inversely associated with patient survival. RBBP4 promoted in vitro TNBC cellular proliferation and in vivo tumor growth but had no effect on invasion or migration. Transcriptomic analyses revealed RBBP4-driven reprogramming of glycolytic metabolism, characterized by Warburg effect-related phenotypes (elevated glucose consumption, lactate generation, and extracellular acidification). At the molecular level, RBBP4 interacted with RelB, activating NF-κB, which led to nuclear RelB translocation and PD-L1 upregulation. Additionally, glycolysis-derived lactate induced H3K18 lactylation, forming a feedforward epigenetic loop that sustained RBBP4 expression. Conclusion: RBBP4 acts as a nodal regulator linking metabolic reprogramming, NF-κB activation, and immune evasion in TNBC. Targeting RBBP4 or its associated downstream pathways may offer viable strategies for managing TNBC. Triple-negative breast cancer RBBP4 Glycolysis NF-κB PD-L1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Breast cancer is a common cancer in women[ 1 ]. The triple-negative breast cancer (TNBC) subtype is highly aggressive and is defined by an absence of estrogen and progesterone receptors, as well as human epidermal growth factor receptor 2 (HER2)[ 2 , 3 ]. This absence prevents the use of conventional targeted or hormone-based therapeutic strategies used for other forms of breast cancer, restricting primary treatment to chemotherapy[ 4 , 5 ]. It is thus important to identify novel biomarkers to guide the stratification and treatment of TNBC patients in the clinic[ 6 ]. If this goal is achieved successfully, this will provide opportunities for more effective individualized therapeutic interventions with the aim of improving TNBC patient outcomes. Retinoblastoma binding protein 4 (RBBP4) controls chromatin remodeling and transcriptional activity, with increasing evidence supporting its involvement in the pathogenesis of several malignancies, including TNBC[ 7 , 8 ]. Functioning as a histone chaperone, RBBP4 belongs to the WD40-repeat protein family and contains a canonical WD40 domain, which adopts a characteristic β-propeller structure. This configuration enables RBBP4 to mediate myriad protein-protein interactions critical for its regulatory roles[ 9 ]. RBBP4 commonly participates in multi-protein chromatin-modifying assemblies such as the nucleosome remodeling and deacetylase (NuRD) complex and the polycomb repressive complex 2 (PRC2). Within these complexes, the WD40 repeats of RBBP4 interact specifically with histone components, including the α1 helix of histone H4 and the N-terminal tail of histone H3, thereby modulating chromatin architecture and influencing gene transcription[ 9 , 10 ]. The disruption of RBBP4 expression or function has been linked to aberrant chromatin remodeling, ultimately leading to dysregulated transcriptional programs that can drive tumor onset and development, and also contribute to the pathophysiology of other diseases[ 9 ]. Notably, dysregulated RBBP4 expression has been documented in several human tumors, including glioblastoma and gastric carcinoma, where its expression correlates with distinct clinicopathological characteristics and may reflect underlying disease severity[ 11 – 13 ]. The NF-κB axis is involved in various processes, including proliferation, metastasis, inflammation, and therapeutic resistance in TNBC and other forms of cancer[ 14 – 16 ]. There are five core NF-κB subunits: RelA (p65), RelB, c-Rel, NF-κB1 (p50), and NF-κB2 (p52)[ 17 , 18 ]. Under basal conditions, these are anchored in the cytoplasm by IκB, thereby preventing their transcriptional activity. Stimulation activates the NF-κB axis through the phosphorylation and ubiquitin-mediated degradation of IκB proteins, including IκBα, resulting in the nuclear translocation of the active p65/p50 dimer. Once in the nucleus, these complexes promote the transcription of genes involved in inflammatory responses, immune modulation, and cell survival[ 18 ]. Evidence shows that constitutive activation of the NF-κB axis is a hallmark of TNBC, suggesting its potential in treating the disease[ 19 ]. RelB occupies a distinctive niche among NF-κB subunits owing to its unique participation in the non-canonical branch of the axis. RelB has been found to contribute to tumorigenesis and modulate cellular responses to anticancer therapies[ 20 ]. Accordingly, targeting RelB and its downstream effectors has gained traction as a potential chemotherapeutic strategy, offering new avenues for potential cancer treatment[ 21 ]. Energy metabolism reprogramming is a hallmark of cancer[ 22 ], including the prominent dysregulation of glucose catabolism[ 23 ]. This form of enhanced glucose utilization by tumor cells through aerobic glycolysis, also termed the Warburg effect[ 24 , 25 ]. Aerobic glycolysis entails higher levels of glucose internalization together with the preferential production of lactate even when oxygen is present, thus helping to generate the energy and biosynthetic products needed to drive metastatic tumor growth and proliferation[ 24 ]. Recent evidence further emphasizes the involvement of novel tumor driver genes in the control of TNBC onset and progression through the regulation of glycolytic metabolism[ 3 , 26 ]. Here, the expression and functions of RBBP4 in TNBC were comprehensively investigated. To achieve this goal, mRNA and protein levels of RBBP4 were assessed in clinical specimens using qPCR and immunohistochemistry (IHC), respectively. These analyses revealed a marked increase in RBBP4 expression within TNBC tissues relative to normal tissue samples. Furthermore, higher RBBP4 levels were significantly correlated with reduced patient survival, suggesting its potential as an indicator of prognosis. To further delineate the functional relevance of RBBP4 in TNBC pathophysiology, cellular and animal experiments were performed, focusing on its influence on cell proliferation, metastatic potential, and metabolic reprogramming. These investigations offer novel mechanistic insights into RBBP4-mediated tumor progression and propose RBBP4 as a candidate molecular target for therapeutic intervention in TNBC. Matierrials and methods Cells and transfections The human TNBC MDA-MB231, MDA-MB468, HS578T, and HCC1937 cell lines were procured from Procell Life Science (Wuhan, China). The cells were authenticated using short tandem repeat profiling, and were routinely screened for mycoplasma using a kit (Beyotime). All cells were grown in DMEM with 10% fetal bovine serum (FBS; Gibco) at 37°C with 5% CO 2 . Cells were transiently transfected using Lipofectamine 2000 (Invitrogen) as directed. For stable overexpression of RBBP4, MDA-MB231 cells were transfected with a pcDNA3.1-RBBP4-3×Flag construct and selected in G418 (400 µg/mL) for two weeks. For gene silencing, HCC1937 cells were transfected with either specific small interfering RNAs (siRNAs) targeting RBBP4 or a scrambled siRNA control (siNC). The silencing efficiency was evaluated after 48 hours by qPCR and Western blotting to confirm optimal conditions. Database analyses Expression levels of RBBP4 in TNBC subgroups were assessed using UALCAN, a web-based analytical tool that leverages data from The Cancer Genome Atlas (TCGA, https://cancergenome.nih.gov/ ) to facilitate the exploration of gene expression and survival outcomes across cancer types[ 27 ]. Additionally, the Xiantao bioinformatics platform ( https://www.xiantao.love/ ), which integrates standardized TCGA datasets, was employed to compare RBBP4 expression in breast cancer tissues versus normal controls, providing complementary insights into its differential expression. Patient samples A total of 120 matched pairs of formalin-fixed, paraffin-embedded (FFPE) TNBC tissues and adjacent non-tumorous tissues were retrospectively collected from resection cases between January 2010 and October 2011. An independent set of 120 freshly frozen breast tumors and adjacent normal tissues was also included. All procedures involving human samples were approved by the ethics committee at the First Affiliated Hospital of Xi’an Jiaotong University, and written informed consent was obtained from all participants. Immunohistochemistry FFPE tissue sections (4 µm thick) were mounted and heated at 60°C for 2 hours, and then deparffinized in xylene and rehydrated using an ethanol gradient. Antigen retrieval was conducted by steaming sections in citrate (pH 6.0) or EDTA (pH 8.0) buffers at 95°C for 10 minutes. The activities of endogenous peroxidases were blocked with 3% H 2 O 2 for 15 minutes. After blocking with 10% BSA (1 hour, ambient temperature), sections were treated overnight with primary antibodies at 4°C. After washing with PBS, the sections were exposed to appropriate secondary antibodies (anti-rabbit or anti-mouse) for 30 minutes, followed by visualization with liquid DAB + substrate (#9018, Golden Bridge, China) and nuclear counterstaining with Carazzi’s hematoxylin (#BL702A, Biosharp, China). Two pathologists independently evaluated all stained slides. qPCR Total RNA was extracted from frozen tissue specimens using TRIzol, and homogenization was performed using a mechanical tissue disruptor. RNA was isolated from cells using the RNA-Quick Purification Kit (Yishan Biotechnology, Shanghai, China). A PrimeScript RT reagent Kit (Takara, Japan) was utilized for reverse-transcription of RNA to cDNA, and qPCR was performed with SYBR Green Master Mix (Cwbio, Beijing, China) on an ABI 7500 PCR platform. GAPDH was the internal reference and relative gene expression was determined using the 2 −ΔΔCt method. Primer sequences are given in Supplementary Table 1. Western blotting and co-immunoprecipitation (co-IP) Whole-cell lysates were prepared with RIPA buffer (Beyotime, Shanghai, China) with protease and phosphatase inhibitors (Cwbio, Beijing, China) and 1 mM PMSF. For cytoplasmic and nuclear protein fractionation, a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) was utilized as directed. For Co-IP assays, cells were lysed in NP-40 buffer with protease and phosphatase inhibitors and lysates were treated overnight with antibodies against RBBP4, IκBα, or HA at 4°C, followed by capture of the complexes with Protein A /G agarose for 3 hours. Following three washes with PBS-T, elution was performed by boiling in SDS loading buffer and resolution with SDS-PAGE before transfer to PVDF membranes, blocking (5% BSA, 1 hour), and probing with appropriate primary and HRP-conjugated secondary antibodies. Bands were visualized using an enhanced chemiluminescence system (Tanon 5200, Shanghai, China). Immunofluorescence (IF) and multiplex immunofluorescence (mIF) For single-color IF, cells were grown on coverslips in 6-well plates for 48 hours and fixed with 4% paraformaldehyde. Following PBS washes, cells were permeabilized and blocked with 5% normal goat serum and 0.3% Triton X-100 for 1 hour. Primary antibodies were applied overnight at 4°C. The next day, Alexa Fluor 488- or 555-conjugated donkey anti-rabbit secondary antibodies were added for 1 hour, nuclei were counterstained with DAPI, and cells were imaged with a Leica TCS SP5 confocal microscope (Wetzlar, Germany). mIF staining was conducted using a four-color tyramide signal amplification (TSA) kit (Absin, #abs50012, China), as per the manufacturer’s protocol. After antigen retrieval and blocking, tissue sections were treated sequentially with primary antibodies for 30 to 60 minutes at 37°C, followed by HRP-conjugated secondary antibodies and TSA reagents conjugated to Opal dyes. After nuclear staining with DAPI, tissues were mounted in glycerol-gelatin medium for fluorescence imaging. Live-cell imaging To monitor cellular dynamics in real time, cells (3 ~ 4×10 4 /well) in 0.8 mL of complete medium were inoculated into 24-well plates. Immediately after seeding, time-lapse imaging was initiated using the zenCell Owl live-cell microscopy system (innoME GmbH, Germany) within a standard tissue culture incubator. The system captured one image per hour across 24 parallel channels. Following a 24-hour adhesion period, experimental treatments were applied, and imaging resumed 3 hours post-treatment, continuing until the cultures reached confluency. Image analysis and quantification of cell proliferation were performed using ZenCell Owl software to generate growth curves over time. Migration and invasion assessments For both invasion and migration assays, Transwell inserts (Corning) were employed. In invasion assays, the upper chambers were pre-coated with Matrigel (Solarbio). Cells suspended in serum-free medium at a density of 2.5×10⁵ cells per mL were seeded into the upper compartments, with medium supplemented with 20% fetal bovine serum (FBS) added to the lower compartments. Following 24 hours of incubation under standard culture conditions, cells remaining on the upper surface of the membrane were removed, and those that had invaded to the lower surface were fixed using the aforementioned method. The Transwell inserts were then transferred to wells containing diluted DAPI solution (1 µg/mL) for 10 minutes of light-protected incubation, followed by three washes in phosphate-buffered saline (PBS). Representative fields of the cells were imaged using a fluorescence microscope to quantify the invaded cells. Migration assays were performed using an identical protocol, excluding the Matrigel coating step. RNA-sequencing Transcriptomic profiling was carried out on RBBP4-overexpressing cells and vector-transfected controls. After rinsing with cold PBS, cells were trypsinized and lysed in TRIzol for total RNA extraction. RNA library construction and high-throughput sequencing were undertaken on an Illumina HiSeq platform by Applied Protein Technology (Beijing, China). Differentially expressed genes (DEGs) were identified using DESeq2 in R, with the criteria of fold change > 1.5 and adjusted p -value < 0.05 (see Supplementary Table 2). Gene Ontology (GO) and KEGG enrichment analyses were undertaken via the DAVID database, with visual representations of enriched biological processes and pathways created using the Goplot R package. Glucose and lactate measurements Cellular glucose uptake was evaluated using a Glucose Oxidase Method Kit (APPLYGEN, China), while lactate generation was quantified using a Lactic Acid Assay Kit (Nanjing Jiancheng Bioengineering Institute, China). For both assays, cells were grown in 6-well plates for 24 hours. Conditioned media were then collected following centrifugation to eliminate cells. The concentration of glucose and lactate in the supernatants was determined as directed. Metabolic assays The metabolic profile of cells, including both the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR), was examined using a Seahorse XF96 Extracellular Flux Analyzer (Agilent Technologies). TNBC cells transfected with RBBP4-targeting siRNA or siNC were inoculated in XF96-well plates at densities of 1×10 5 /well. Following an 18-hour incubation, the media were replaced with Seahorse XF RPMI (pH 7.4) with 10 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate. Cells were equilibrated in a non-CO₂ incubator for one hour prior to analysis. OCR and ECAR values were recorded in real-time upon sequential injection of metabolic modulators, as per the provided protocol. Following completion of measurements, cells were stained with Hoechst 33342 (Solarbio), imaged using the ImageXpressMicro Confocal system (Molecular Devices), and quantified for normalization purposes. Results were analyzed with Seahorse Wave Pro software (Agilent Technologies). ATP generation was assessed using the Seahorse ATP Rate Assay Kit (Agilent Technologies), involving sequential introduction of oligomycin (1.5 µM) and a rotenone/antimycin A mixture (0.5 µM each). Mitochondrial respiratory function was determined via the Mito Stress Test Kit, using the same concentrations of oligomycin, FCCP (2 µM), and rotenone/antimycin A. Glycolytic capacity was measured with a Glycolytic Rate Assay Kit, following treatment with rotenone/antimycin A, 2-deoxyglucose (2-DG, 50 mM), and vehicle. For glycolytic stress testing, cells were incubated in medium containing only L-glutamine (2 mM) and sequentially treated with D-glucose (10 mM), oligomycin (1 µM), and 2-DG (50 mM). All assays were performed using media containing glucose, glutamine, and pyruvate unless otherwise specified. In vivo xenograft tumor assays Twelve BALB/c nude mice (female, six weeks old) were randomly assigned to two groups. Animals were given subcutaneous injections of 5×10 6 HCC1937 cells either stably overexpressing RBBP4 or carrying the control vector, administered into the right hypochondriac region. Tumor formation was monitored beginning one week post-injection, with tumor volumes measured biweekly using the standard formula: ½ × L × W² (where L = length and W = width). After 24 days, mice were euthanized, and the tumors were collected, imaged, and stained with hematoxylin and eosin (HE) or IHC. All animal experiments were reviewed and approved by the l Animal Care and Use Committee of the Medical College of Xi'an Jiaotong University . Statistical analyses All statistical analyses were conducted using SPSS 19.0 and GraphPad Prism 7. Data are shown as the mean ± standard deviation from a minimum of 3 independent biological replicates. For comparison between two groups, an independent samples t-test was used when data met normality assumptions; otherwise, the non-parametric rank sum test was applied. One-way ANOVAs were used for comparisons involving more than two groups. Fisher’s exact test was employed for analyzing associations between categorical clinicopathological variables. A p -value less than 0.05 was deemed significant. Results RBBP4 upregulation is evident in TNBC and linked to poor prognostic outcomes in patients To explore the clinical relevance of RBBP4 in TNBC, its expression was assessed utilizing the UALCAN database. As illustrated in Fig. 1 A and 1 B and Supplementary Figure S1 A, RBBP4 transcript levels were significantly elevated in TNBC tissue samples relative to normal tissues (P < 0.05). However, survival analysis using the Xiantao database did not reveal a statistically significant difference in patient outcomes based on RBBP4 expression levels (Supplementary Fig. S1 B). Given the limitations of the Xiantao dataset as a means of distinguishing TNBC from other breast cancer subtypes, we proceeded with direct analysis of clinical specimens. Next, qPCR and IHC approaches were utilized to examine RBBP4 mRNA and protein levels, respectively, in tumor and matched normal samples from 120 TNBC cases who underwent surgical resection between September 2010 and December 2011. Both assays demonstrated a marked upregulation of RBBP4 in tumor tissues relative to adjacent non-tumor tissues (P < 0.01; Fig. 1 C and 1 D). Kaplan–Meier curves indicated that high intratumoral RBBP4 levels were linked with reduced overall survival (P = 0.0292; Fig. 1 E). Furthermore, Western blotting of TNBC cells showed that RBBP4 protein levels were markedly raised in HCC1937 cells relative to MDA-MB231 cells (P < 0.05; Fig. 1 F). Collectively, these results indicate that RBBP4 is markedly overexpressed in TNBC and may thus represent a prognostic indicator for this cancer type. RBBP4 promotes in vitro TNBC cell proliferation without impacting their metastatic potential To examine the functions of RBBP4 in TNBC progression, we engineered MDA-MB231 and HCC1937 cell lines to either overexpress or silence RBBP4 using plasmid transfection and siRNA, respectively. Quantitative PCR confirmed significant changes in RBBP4 transcript levels following transfection in both models (P < 0.01; Fig. 2 A), and these alterations were validated at the protein level via Western blotting (Fig. 2 B). HCC1937 cells were used for knockdown experiments, while MDA-MB231 cells were employed for overexpression studies. Proliferation was monitored using a zenCELL Owl live-cell imaging platform, capturing hourly images over a 96-hour period. Normalized growth curves and doubling time analyses demonstrated a pronounced proliferative advantage in RBBP4-overexpressing cells compared to controls (Fig. 2 C). This finding was further supported by EdU incorporation and colony formation assays, which revealed that RBBP4 knockdown markedly decreased proliferation in HCC1937 cells, while its overexpression enhanced proliferation in MDA-MB231 cells (P < 0.01; Fig. 2 D and 2 E). To investigate the potential effect of RBBP4 on cellular motility, we performed wound healing and Transwell assays. These experiments revealed no significant alterations in either migration or invasion following RBBP4 overexpression or knockdown (Supplementary Figs. S2 and S3). These results suggest that while RBBP4 facilitates TNBC cell proliferation, it does not modulate metastatic behaviors in vitro . RBBP4 enhances in vivo TNBC tumor growth To clarify the effects of RBBP4 on tumor growth in a xenograft model, nude mice were injected with MDA-MB231 cells stably overexpressing RBBP4 or with vector control cells. Biweekly tumor volume measurements indicated that RBBP4-overexpressing cells produced significantly larger tumors over time (P < 0.01; Fig. 3 A, B). At the study endpoint, tumors in the RBBP4-overexpressing group exhibited significantly greater weights compared to controls (P < 0.01; Fig. 3 C). Histopathological examination confirmed that no liver metastases were detectable in either group (Fig. 3 D). IHC analysis of excised tumors showed markedly increased staining for both RBBP4 and the proliferation marker Ki67 in the overexpression group relative to controls (Fig. 3 E). These findings confirm that RBBP4 enhances TNBC tumor growth in vivo . Glycolytic activity driven by RBBP4 facilitates the proliferation of TNBC cells RNA sequencing of RBBP4- overexpressing MDA-MB231 cells identified 93 protein-coding DEGs, including 56 upregulated and 37 downregulated transcripts (Fig. 4 A; Supplementary Table 2). Functional enrichment analyses using KEGG and GO databases revealed that several pathways involved in metabolic regulation were significantly affected (Fig. 4 B; Supplementary Figs. S4A, S4B). Notably, the upregulated genes CDC25A and JUN, both of which are implicated in glycolytic activation, were prominently enriched in the RBBP4-overexpressing group. Investigation of glucose uptake and lactate secretion in RBBP4-manipulated cells showed that both parameters were markedly elevated upon RBBP4 overexpression (Fig. 4 C, D). To further test the dependency of RBBP4-driven proliferation on glycolytic flux, we conducted colony formation assays using 2-DG, a competitive glycolytic inhibitor, and D-galactose, which slows glycolysis due to its delayed entry into the pathway. Both treatments markedly suppressed the pro-proliferative effects induced by RBBP4 overexpression (Fig. 4 E). To quantify the metabolic reprogramming more precisely, the ECAR and OCR were determined using Seahorse metabolic analysis. ECAR, an indicator of glycolytic activity, was significantly elevated in RBBP4-overexpressing cells (Fig. 4 F, G), while OCR, a measure of oxidative phosphorylation, remained unchanged (Supplementary Figs. S5A, S5B). These findings support a model in which RBBP4 enhances glycolytic flux in a manner consistent with the induction of the Warburg effect, thereby promoting TNBC cell proliferation. RBBP4 modulates NF-κB activation and PD-L1 levels To further explore the downstream molecular pathways regulated by RBBP4, we performed KEGG enrichment analysis and GSEA on transcriptomic data from MDA-MB231 cells with RBBP4 overexpression. These analyses revealed significant upregulation of genes associated with the NF-κB signaling cascade (Fig. 4 B and Fig. 5 A). Given the established role that NF-κB plays in TNBC pathogenesis, we hypothesized that RBBP4 may enhance tumor progression via activation of this pathway. Among the analyzed members of the NF-κB family, RelB exhibited the most substantial expression change in this experimental context. To determine whether RBBP4 influences RelB activation, we assessed its subcellular localization via nuclear-cytoplasmic fractionation and Western blotting. RBBP4 overexpression promoted nuclear translocation of RelB, accompanied by decreased cytoplasmic retention (Fig. 5 B). Co-IP assays demonstrated a physical interaction between RBBP4 and RelB (Fig. 5 C), and immunofluorescence confirmed their co-localization in the nucleus (Fig. 5 D). As NF-κB activation has been linked to PD-L1 upregulation, we next assessed the relationship between RBBP4 and CD274 (PD-L1) levels utilizing information from the Xiantao database[ 28 ]. This showed a marked positive association between RBBP4 and PD-L1 (R = 0.156, P = 0.002; Fig. 5 E). Since RelB is a transcription factor capable of interacting with the promoter regions of target genes and has previously been shown to interact with the PD-L1 promoter[ 29 ], we conducted chromatin immunoprecipitation (ChIP) followed by PCR to validate this interaction. Our ChIP-PCR results confirmed direct binding between RELB and the PD-L1 promoter (Fig. 5 F). To further support this finding, multiplex immunofluorescence analysis of TNBC tissue samples revealed that both RelB and PD-L1 levels were markedly elevated in specimens exhibiting high RBBP4 expression (Fig. 5 G). We then employed siRNA-mediated knockdown of RelB to assess its functional relevance. A reduction in RelB expression led to decreased PD-L1 levels, indicating a regulatory relationship. Moreover, colony formation assays indicated that RelB knockdown impaired the clonogenic capacity of TNBC cells (Fig. 5 J). These findings thus suggest that RBBP4 raises PD-L1 levels by NF-κB activation through modulation of RelB activity, thereby contributing to TNBC progression. Lactate-induced H3K18la-mediated upregulation of RBBP4 is evident in TNBC Emerging evidence suggests that lactate, a metabolic byproduct of glycolysis, can drive protein lactylation, a post-translational modification that influences stability and gene expression. H3K18la lactylation has been the most thoroughly studied form of such modification[ 30 , 31 ]. Western blotting showed markedly raised H3K18la levels in TNBC tissues relative to controls, indicating that histone lactylation is a frequent event in TNBC (Fig. 6 A). To determine whether lactylation regulates RBBP4 expression, we exposed HCC1937 cells to varying lactate concentrations, indicating dose-dependent increases in RBBP4 contents in response to lactate (Fig. 6 B). Conversely, treatment with 2-DG or oxamate, an LDHA inhibitor, led to a marked suppression of RBBP4 expression in a dose-dependent manner (Fig. 6 C, 6 D). Notably, oxamate also decreased H3K18la levels, while co-treatment with sodium lactate (Nala) reversed oxamate effects on both RBBP4 and H3K18la level (Fig. 6 E). To further examine whether RBBP4 expression is directly regulated by H3K18la, we performed ChIP-qPCR assays in which H3K18la was enriched at the RBBP4 promoter region, with this enrichment having been significantly diminished following treatment with 2-DG or oxamate (Fig. 6 F, 6 G). These findings indicate that lactate produced via glycolytic activity can enhance RBBP4 transcription in TNBC cells through H3K18la-dependent epigenetic regulatory mechanisms. Discussion Dysregulated expression of RBBP4 is commonly associated with aberrant chromatin remodeling and disrupted transcriptional[ 9 ]. Our study reveals a multifaceted role for RBBP4 in TNBC, linking its elevated expression to poor clinical outcomes and enhanced tumor aggressiveness. Analysis of the UALCAN data indicated marked upregulation of RBBP4 in TNBC tissues relative to normal counterparts, suggesting its potential utility as a diagnostic biomarker. This initial finding was corroborated by qPCR and immunohistochemistry analyses performed on clinical specimens from 120 TNBC patients, which further demonstrated that elevated RBBP4 levels were associated with significantly reduced survival, positioning RBBP4 as a candidate prognostic biomarker in this cancer type. Uncontrolled proliferation is a hallmark of cancer and is linked to both disease progression and poor clinical outcomes[ 32 ]. Understanding the molecular regulators of cell division is thus essential for identifying therapeutic targets[ 33 ]. In this context, our in vitro assays identified RBBP4 as a promoter of TNBC cell proliferation. Specifically, siRNA-mediated knockdown of RBBP4 in HCC1937 cells led to substantial suppression of proliferation, whereas forced overexpression in MDA-MB231 cells enhanced cell growth. These functional effects were validated through live-cell imaging, EdU incorporation, and colony formation assays. Notably, RBBP4 manipulation had no observable effect on migratory or invasive behaviors in vitro, suggesting that its oncogenic role is primarily confined to promoting cellular proliferation rather than metastasis. To verify the cellular findings, we generated mouse xenograft model using MDA-MB231 cells engineered to overexpress RBBP4. Tumors derived from RBBP4-overexpressing cells exhibited significantly accelerated growth and increased mass relative to control tumors. Histopathological analysis confirmed increased proliferative activity, as indicated by enhanced Ki-67 staining and higher RBBP4 expression. These results, taken together with our in vitro observations, establish RBBP4 as a critical driver of TNBC proliferation, both in culture and in vivo, highlighting its potential in treating the disease. The dysregulation of glycolysis is associated with sustaining rapid tumor cell growth and survival under hypoxic conditions[ 34 ]. In addition to its importance as an energy source, glycolysis also facilitates oncogenic signaling and biosynthetic pathways[ 35 ]. Prior studies have identified glycolytic enzymes such as PKM2 and PFKFB3 as oncogenic drivers[ 35 ]. For instance, PKM2 overexpression is linked with adverse clinical outcomes and transcriptional activation in various cancers[ 36 ]. Similarly, inhibition of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) can lead to the inhibition of angiogenic activity[ 37 ]. Given the centrality of glycolysis to TNBC aggression, RBBP4 likely shapes tumor progression through effects on these mechanisms. NF-κB signaling is also central to progressive tumor growth[ 38 ], driving oncogenesis through the activation of genes that favor proliferative, migratory, and invasive activity[ 39 ]. NF-κB controls glycolytic enzymes at the transcriptional level[ 34 ]. Tumor-associated macrophage (TAM)-derived IL-1α can induce NF-κB activation in TNBC, resulting in enhanced glycolytic activity and cytokine biogenesis[ 40 ]. RNA-seq analyses and functional enrichment assays unveiled the effects of RBBP4 overexpression on glycolysis and other metabolic pathways, as further evidenced by elevated ECAR levels and higher levels of glucose consumption in these RBBP4-overexpressing cells, triggering the Warburg effect. Such metabolic reprogramming is crucial for the highly proliferative demands of TNBC cells[ 41 ]. The NF-κB pathway was also identified herein as a crucial downstream effector of RBBP4, as its overexpression resulted in NF-κB activation characterized by RelB nuclear translocation and interaction with RBBP4. RelB interaction with the PD-L1 promoter was also noted, consistent with NF-κB-mediated modulation of PD-L1 levels and the consequent ability of tumor cells to establish an immunosuppressive microenvironment. RelB binding to the PD-L1 promoter and consequent PD-L1 upregulation may be indicative of the role of RBBP4 as a modulator of TNBC immune responses through the control of PD-L1 expression, allowing tumors to evade immune-mediated destruction. As a glycolysis byproduct, lactate can facilitate lysine lactylation on histones, thereby inducing transcriptional activity[ 42 ]. H3K18 lactylation and other forms of this epigenetic modification have recently been linked to oncogenic development, progressive tumor growth, metabolic reprogramming, and immune evasion[ 43 , 44 ]. In the present study, elevated H3K18 lactylation (H3K18la) levels were noted in TNBC tissue samples, consistent with the prevalence of this modification in this form of cancer. The upregulation of RBBP4 was further noted in response to lactate treatment, whereas it was downregulated in response to the glycolytic inhibitors oxamate and 2DG. Strikingly, the application of the lactate analog Nala resulted in the reversal of oxamate-mediated suppression of RBBP4 and H3K18la levels. ChIP and qPCR results provided further confirmation of H3K18la enrichment at the RBBP4 promoter, with both oxamate and 2DG partially abrogating this enrichment. 2DG and oxamate. Together, these results offer evidence that lactate-mediated H3K18la recruitment to the RBBP4 promoter can lead to the enhanced expression of RBBP4 (Fig. 7 ). Conclusions The study findings highlight RBBP4 as a key regulator of progressive TNBC tumor growth attributable to its ability to modulate metabolic activity, immune functionality, and cellular proliferation. As TNBC tissue samples exhibited elevated RBBP4 levels that were associated with poor patient outcomes, these findings underscore the potential of RBBP4 as a marker and target for the disease. However, future studies exploring the precise mechanisms whereby RBBP4 shapes the progression of TNBC will be essential, as will efforts to validate its performance as a therapeutic target in a clinical context. Abbreviations triple-negative breast cancer (TNBC); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H3K18la, H3 lysine 18 lactylation; IgG, immunoglobulin G; ChIP-qPCR, chromatin immunoprecipitation-quantitative polymerase chain reaction; 2DG, 2-deoxyglucose. Declarations Consent for publication Each author approved the manuscript before submission for publication. Ethics approval and consent to participate The studies involving humans were approved by the ethical committees at the First Affiliated Hospital of Xi’an Jiaotong University. The studies were conducted in accordance with the local legislation and institutional requirements, and we have obtained written informed consent from all participants involved in the study. The animal studies were approved by the Medical College of Xi'an Jiaotong University. The studies were conducted in accordance with the local legislation and institutional requirements. Availability of data and materials All data generated or analysed during this study are included in this published article [and its supplementary information files]. Competing interests The authors declare that they have no competing interests. Funding This work was supported by grants from the Natural Science Foundation of Shaanxi Province (2019JM-115, 2018JQ8030, 2025SF-YBXM-385). Author contributions XD: Conceptualization, Data curation, Investigation, Methodology, Visualization, Writing – original draft. HX: Investigation, Methodology, Visualization, Writing – original draft. PZ: Investigation, Methodology, Visualization, Writing – original draft. JL: Conceptualization, Investigation, Funding acquisition, Writing – original draft. SS; Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing –review & editing. All authors read and approved the final manuscript. References Filho AM, Laversanne M, Ferlay J, Colombet M, Pieros M, Znaor A, Parkin DM, Soerjomataram I, Bray F. The GLOBOCAN 2022 cancer estimates: Data sources, methods, and a snapshot of the cancer burden worldwide. INTERNATIONAL JOURNAL OF CANCER. 2025;156. Ligorio F, Vingiani A, Torelli T, Sposetti C, Drufuca L, Iannelli F, Zanenga L, Depretto C, Folli S, Scaperrotta G, et al. Early downmodulation of tumor glycolysis predicts response to fasting-mimicking diet in triple-negative breast cancer patients. Cell Metab. 2025;37:330-44.e7. Zhao G, Liu Y, Yin S, Cao R, Zhao Q, Fu Y, Du Y. FOSL1 transcriptionally dictates the Warburg effect and enhances chemoresistance in triple-negative breast cancer. J Transl Med. 2025;23:1. Leon-Ferre RA, Goetz MP. 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J Transl Med. 2025;23:247. Hanahan D. Hallmarks of Cancer: New Dimensions. Cancer Discov. 2022;12:31-46. Feitelson MA, Arzumanyan A, Kulathinal RJ, Blain SW, Holcombe RF, Mahajna J, Marino M, Martinez-Chantar ML, Nawroth R, Sanchez-Garcia I, et al. Sustained proliferation in cancer: Mechanisms and novel therapeutic targets. Semin Cancer Biol. 2015;35 Suppl:S25-25S54. Chelakkot C, Chelakkot VS, Shin Y, Song K. Modulating Glycolysis to Improve Cancer Therapy. Int J Mol Sci. 2023;24:2606. Yang J, Wang H, Li B, Liu J, Zhang X, Wang Y, Peng J, Gao L, Wang X, Hu S, et al. Inhibition of ACSS2 triggers glycolysis inhibition and nuclear translocation to activate SIRT1/ATG5/ATG2B deacetylation axis, promoting autophagy and reducing malignancy and chemoresistance in ovarian cancer. Metabolism. 2025;162:156041. Yu S, Zang W, Qiu Y, Liao L, Zheng X. Deubiquitinase OTUB2 exacerbates the progression of colorectal cancer by promoting PKM2 activity and glycolysis. Oncogene. 2022;41:46-56. Icard P, Alifano M, Simula L. Citrate oscillations during cell cycle are a targetable vulnerability in cancer cells. Biochim Biophys Acta Rev Cancer. 2025;1880:189313. Kumar N, Sethi G. Telomerase and hallmarks of cancer: An intricate interplay governing cancer cell evolution. Cancer Lett. 2023;578:216459. Devis-Jauregui L, Eritja N, Davis ML, Matias-Guiu X, Llobet-Navàs D. Autophagy in the physiological endometrium and cancer. Autophagy. 2021;17:1077-95. De Paolis V, Troisi V, Bordin A, Pagano F, Caputo V, Parisi C. Unconventional p65/p52 NF-κB module regulates key tumor microenvironment-related genes in breast tumor-associated macrophages (TAMs). Life Sci. 2024;357:123059. Wang Z, Jiang Q, Dong C. Metabolic reprogramming in triple-negative breast cancer. Cancer Biol Med. 2020;17:44-59. Peng X, Du J. Histone and non-histone lactylation: molecular mechanisms, biological functions, diseases, and therapeutic targets. Molecular Biomedicine. 2025;6. Li W, Zhou C, Yu L, Hou Z, Liu H, Kong L, Xu Y, He J, Lan J, Ou Q, et al. Tumor-derived lactate promotes resistance to bevacizumab treatment by facilitating autophagy enhancer protein RUBCNL expression through histone H3 lysine 18 lactylation (H3K18la) in colorectal cancer. Autophagy. 2024;20:114-30. Yang Y, Luo N, Gong Z, Zhou W, Ku Y, Chen Y. Lactate and lysine lactylation of histone regulate transcription in cancer. Heliyon. 2024;10:e38426. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-7280626","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":497714055,"identity":"f6c2313b-0b88-4959-8365-fc982436df56","order_by":0,"name":"Xuyuan Dong","email":"","orcid":"","institution":"The First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Xuyuan","middleName":"","lastName":"Dong","suffix":""},{"id":497714056,"identity":"8a430cad-23de-45f0-b251-db349ab7e025","order_by":1,"name":"Hongyan Xu","email":"","orcid":"","institution":"The First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Hongyan","middleName":"","lastName":"Xu","suffix":""},{"id":497714057,"identity":"c48dbe96-3c5d-4818-8e9a-2d3810f0c700","order_by":2,"name":"Pengcheng zou","email":"","orcid":"","institution":"The First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Pengcheng","middleName":"","lastName":"zou","suffix":""},{"id":497714058,"identity":"83c899f9-df1e-47ab-8d65-0b2bf6683dc8","order_by":3,"name":"Jianun Lei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIie2Pv0rEQBCHJyxsmrlb7CbkJQaEO8Id56usBLSzEQ6rMxCIpS9wD3GHYL2yxXXWghaxSWVhFSzNGsE/sNHyiv2KmR/DfMwuQCCwh8RlVwhAgxDGBcX9wE/0pUgNpCHZ/KkUfdcAyK79Q7kSTZ1VT1rF2FL2ZmkK4u4RYXHmf5icclI1OilHN0zaUlbIfIaQnw/8ZUJJZTXb0bbulBUbnKQI5rjwKnH7oRxZrI27wka1g8pBiZ9XBEZ1r6AcVFKBS6J7q8nKQ6aTU+IuZGvOvcpY7W5TWtpLdW2blOYz4l35/PBysfAqDkE/gnCFB/Y7otffIRAIBALfeQeIRU1CiXhDtwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-8258-705X","institution":"Xi'an Jiaotong University Medical College First Affiliated Hospital: The First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":true,"prefix":"","firstName":"Jianun","middleName":"","lastName":"Lei","suffix":""},{"id":497714059,"identity":"0a7220b4-79f9-4381-b9b9-6a0401e1d1c8","order_by":4,"name":"Shan Shao","email":"","orcid":"","institution":"The First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Shan","middleName":"","lastName":"Shao","suffix":""}],"badges":[],"createdAt":"2025-08-03 00:28:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7280626/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7280626/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89232883,"identity":"a403492f-d4d2-4ae7-94d6-94781b1809ea","added_by":"auto","created_at":"2025-08-17 14:30:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1619367,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of RBBP4 expression in TNBC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) RBBP4 expression levels in breast cancer subtypes, using data from the UALCAN database. (B) RBBP4 expression profiles in specific subtypes of breast invasive carcinoma (BRCA), with a focus on TNBC. (C) qPCR measurement of \u003cem\u003eRBBP4\u003c/em\u003emRNA levels in 120 pairs of TNBC tumor and paracancerous tissue samples. (D) Immunohistochemical (IHC) staining for RBBP4 in TNBC tissue samples, Scale bar: 200 μm. (E) Kaplan-Meier curves of the correlation between RBBP4 levels and TNBC patient prognosis. (F) Western blots showing RBBP4 level in MDA-MB-231, MDA-MB-468, HS578T, and HCC1937 cells. Data are means ± standard deviation (SD). *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7280626/v1/c5247a7c80a6e314996bc9c4.png"},{"id":89231723,"identity":"b03cf6cc-68f1-4e86-acfb-da37def6cbb6","added_by":"auto","created_at":"2025-08-17 14:22:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1453988,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSilencing RBBP4 inhibits TNBC cell proliferation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) qPCR measurement of RBBP4 levels in TNBC cells after siRNA-mediated knockdown or plasmid-mediated overexpression. (B) Western blotting evaluation of RBBP4 protein levels in TNBC cells following its siRNA-mediated silencing or plasmid-induced overexpression. (C) zenCELL Owl microscopy-based live-cell imaging of TNBC cells in which RBBP4 had been knocked down or overexpressed. (D) EdU-based analysis of the proliferation of TNBC cells in which RBBP4 had been knocked down or overexpressed, with corresponding quantification. (E) Colony formation by TNBC cells with corresponding quantification. Data are means ± SD; *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7280626/v1/4181bd2ccce31b7466a89ec9.png"},{"id":89230404,"identity":"c4b2a630-7ffe-473a-b689-1bfc44edde8f","added_by":"auto","created_at":"2025-08-17 14:14:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4596740,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRBBP4 silencing inhibits growth of TNBC xenograft tumors.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative xenograft tumor images; Scale bar: 1 cm. (B) Growth of tumors over time. (C) Xenograft tumor weight at the time of sample collection. (D) H\u0026amp;E staining; Scale bar: 200 μm. (E) IHC staining for Ki67 and RBBP4 in xenograft tumor samples; Scale bar: 200 μm. The proportions of positive staining are presented in the right panels. *P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7280626/v1/2167bdef6ab7ce78f69125f5.png"},{"id":89232884,"identity":"c981b86a-b5a1-42d9-8362-3188fd7158cf","added_by":"auto","created_at":"2025-08-17 14:30:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":837069,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRBBP4 enhances glycolytic activity to drive TNBC cell proliferative growth.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Volcano plot of upregulated (red) and downregulated (blue) genes following the introduction of a control or RBBP4 overexpression vector. (B) Top 20 DEG-enriched KEGG pathways when comparing control cells to those overexpressing RBBP4. (C, D) Relative glucose consumption (C) and lactate generation (D) in control cells and cells overexpressing RBBP4. (E) Evaluation of the colony formation activity of cells overexpressing RBBP4 following galactose or 2-DG treatment. (F) Analyses of the glycolytic activity and mitochondrial stress levels in cells in which RBBP4 was overexpressed as determined based on the ECAR. (G) Quantitative analysis of glycolysis, glycolytic capacity, and glycolytic reserve based on the data in (F). 2-DG, 2-deoxyglucose. **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7280626/v1/f076edbda0cfe55b6d3721b7.png"},{"id":89230408,"identity":"ccfa014d-8785-4101-a9a8-a75ed9d05aef","added_by":"auto","created_at":"2025-08-17 14:14:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2088352,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRBBP4 influences NF-κB activation and PD-L1 levels.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) GSEA of the NF-κB axis in the samples with RBBP4 overexpression. (B) Cytoplasmic and nuclear RelB accumulation were assessed in cells overexpressing RBBP4 via Western blotting, with GAPDH and Lamin B as respective normalization controls. (C) Co-immunoprecipitation-based analysis of RBBP4 interactions with RelB. (D) Laser confocal microscopy analysis of subcellular RBBP4 and RelB localization in cells overexpressing RBBP4; Scale bar: 10 μm. (E) Associations between the RBBP4 and CD274 levels performed with the Xiantao database (R = 0.156, P = 0.002). (F) ChIP-PCR analysis of RelB interaction with the CD274 promoter. (G) Multiplex immunofluorescence analysis of RelB and PD-L1 co-localization in groups exhibiting low and high levels of RBBP4 expression; Scale bar: 50 μm. (H) Western blots showing RelB downregulation and/or PD-L1 overexpression. (J) Colony formation assay results assessing the proliferative impact of downregulating RelB and/or overexpressing PD-L1.N=3.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7280626/v1/701793628b9c3fe4c91b7a5d.png"},{"id":89230409,"identity":"eb5b7f05-14ef-4ad0-8a72-78a3cc93ac3a","added_by":"auto","created_at":"2025-08-17 14:14:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":608392,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLactate-induced H3K18la-mediated RBBP4 upregulation is evident in TNBC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blots showing the levels of H3K18la expression in TNBC and normal tissue samples. (B-D) Western blots showing RBBP4 levels in HCC1937 cells. (E) Western blots showing RBBP4 and H3K18la levels in HCC1937 cells with Oxamate or/and Nala. (F, G) ChIP-PCR analysis of H3K18la enrichment on the RBBP4 promoter. **P \u0026lt; 0.01 vs. IgG or H3K18la group.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-7280626/v1/a4810c51904527023953a83e.png"},{"id":89231729,"identity":"3dce3c7b-7610-4884-a816-fcc852f8f640","added_by":"auto","created_at":"2025-08-17 14:22:23","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration of the molecular mechanism of RBBP4 in TNBC.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"placeholderimage.png","url":"https://assets-eu.researchsquare.com/files/rs-7280626/v1/af5544416b10fb9a0ce2d928.png"},{"id":89957034,"identity":"00eb9979-1a96-45dd-9a7c-75e116884c89","added_by":"auto","created_at":"2025-08-26 22:38:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11714356,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7280626/v1/79939f56-e5e3-4054-9347-2ad8d2f88d62.pdf"},{"id":89233560,"identity":"7eaf4fb4-d520-4d6f-9ee6-7b8087a7dd6f","added_by":"auto","created_at":"2025-08-17 14:38:27","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3377850,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7280626/v1/c0140487d752360721bd734c.docx"}],"financialInterests":"","formattedTitle":"RBBP4 orchestrates glycolytic reprogramming and NF-κB-mediated immune evasion in triple-negative breast cancer","fulltext":[{"header":"Background","content":"\u003cp\u003eBreast cancer is a common cancer in women[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The triple-negative breast cancer (TNBC) subtype is highly aggressive and is defined by an absence of estrogen and progesterone receptors, as well as human epidermal growth factor receptor 2 (HER2)[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This absence prevents the use of conventional targeted or hormone-based therapeutic strategies used for other forms of breast cancer, restricting primary treatment to chemotherapy[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. It is thus important to identify novel biomarkers to guide the stratification and treatment of TNBC patients in the clinic[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. If this goal is achieved successfully, this will provide opportunities for more effective individualized therapeutic interventions with the aim of improving TNBC patient outcomes.\u003c/p\u003e\u003cp\u003eRetinoblastoma binding protein 4 (RBBP4) controls chromatin remodeling and transcriptional activity, with increasing evidence supporting its involvement in the pathogenesis of several malignancies, including TNBC[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Functioning as a histone chaperone, RBBP4 belongs to the WD40-repeat protein family and contains a canonical WD40 domain, which adopts a characteristic β-propeller structure. This configuration enables RBBP4 to mediate myriad protein-protein interactions critical for its regulatory roles[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. RBBP4 commonly participates in multi-protein chromatin-modifying assemblies such as the nucleosome remodeling and deacetylase (NuRD) complex and the polycomb repressive complex 2 (PRC2). Within these complexes, the WD40 repeats of RBBP4 interact specifically with histone components, including the α1 helix of histone H4 and the N-terminal tail of histone H3, thereby modulating chromatin architecture and influencing gene transcription[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The disruption of RBBP4 expression or function has been linked to aberrant chromatin remodeling, ultimately leading to dysregulated transcriptional programs that can drive tumor onset and development, and also contribute to the pathophysiology of other diseases[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Notably, dysregulated RBBP4 expression has been documented in several human tumors, including glioblastoma and gastric carcinoma, where its expression correlates with distinct clinicopathological characteristics and may reflect underlying disease severity[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe NF-κB axis is involved in various processes, including proliferation, metastasis, inflammation, and therapeutic resistance in TNBC and other forms of cancer[\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. There are five core NF-κB subunits: RelA (p65), RelB, c-Rel, NF-κB1 (p50), and NF-κB2 (p52)[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Under basal conditions, these are anchored in the cytoplasm by IκB, thereby preventing their transcriptional activity. Stimulation activates the NF-κB axis through the phosphorylation and ubiquitin-mediated degradation of IκB proteins, including IκBα, resulting in the nuclear translocation of the active p65/p50 dimer. Once in the nucleus, these complexes promote the transcription of genes involved in inflammatory responses, immune modulation, and cell survival[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Evidence shows that constitutive activation of the NF-κB axis is a hallmark of TNBC, suggesting its potential in treating the disease[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. RelB occupies a distinctive niche among NF-κB subunits owing to its unique participation in the non-canonical branch of the axis. RelB has been found to contribute to tumorigenesis and modulate cellular responses to anticancer therapies[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Accordingly, targeting RelB and its downstream effectors has gained traction as a potential chemotherapeutic strategy, offering new avenues for potential cancer treatment[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eEnergy metabolism reprogramming is a hallmark of cancer[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], including the prominent dysregulation of glucose catabolism[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This form of enhanced glucose utilization by tumor cells through aerobic glycolysis, also termed the Warburg effect[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Aerobic glycolysis entails higher levels of glucose internalization together with the preferential production of lactate even when oxygen is present, thus helping to generate the energy and biosynthetic products needed to drive metastatic tumor growth and proliferation[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Recent evidence further emphasizes the involvement of novel tumor driver genes in the control of TNBC onset and progression through the regulation of glycolytic metabolism[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHere, the expression and functions of RBBP4 in TNBC were comprehensively investigated. To achieve this goal, mRNA and protein levels of RBBP4 were assessed in clinical specimens using qPCR and immunohistochemistry (IHC), respectively. These analyses revealed a marked increase in RBBP4 expression within TNBC tissues relative to normal tissue samples. Furthermore, higher RBBP4 levels were significantly correlated with reduced patient survival, suggesting its potential as an indicator of prognosis. To further delineate the functional relevance of RBBP4 in TNBC pathophysiology, cellular and animal experiments were performed, focusing on its influence on cell proliferation, metastatic potential, and metabolic reprogramming. These investigations offer novel mechanistic insights into RBBP4-mediated tumor progression and propose RBBP4 as a candidate molecular target for therapeutic intervention in TNBC.\u003c/p\u003e"},{"header":"Matierrials and methods","content":"\u003cp\u003e\u003cb\u003eCells and transfections\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe human TNBC MDA-MB231, MDA-MB468, HS578T, and HCC1937 cell lines were procured from Procell Life Science (Wuhan, China). The cells were authenticated using short tandem repeat profiling, and were routinely screened for mycoplasma using a kit (Beyotime). All cells were grown in DMEM with 10% fetal bovine serum (FBS; Gibco) at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were transiently transfected using Lipofectamine 2000 (Invitrogen) as directed. For stable overexpression of RBBP4, MDA-MB231 cells were transfected with a pcDNA3.1-RBBP4-3\u0026times;Flag construct and selected in G418 (400 \u0026micro;g/mL) for two weeks. For gene silencing, HCC1937 cells were transfected with either specific small interfering RNAs (siRNAs) targeting RBBP4 or a scrambled siRNA control (siNC). The silencing efficiency was evaluated after 48 hours by qPCR and Western blotting to confirm optimal conditions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDatabase analyses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eExpression levels of RBBP4 in TNBC subgroups were assessed using UALCAN, a web-based analytical tool that leverages data from The Cancer Genome Atlas (TCGA, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cancergenome.nih.gov/\u003c/span\u003e\u003cspan address=\"https://cancergenome.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to facilitate the exploration of gene expression and survival outcomes across cancer types[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Additionally, the Xiantao bioinformatics platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.xiantao.love/\u003c/span\u003e\u003cspan address=\"https://www.xiantao.love/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which integrates standardized TCGA datasets, was employed to compare RBBP4 expression in breast cancer tissues versus normal controls, providing complementary insights into its differential expression.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePatient samples\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 120 matched pairs of formalin-fixed, paraffin-embedded (FFPE) TNBC tissues and adjacent non-tumorous tissues were retrospectively collected from resection cases between January 2010 and October 2011. An independent set of 120 freshly frozen breast tumors and adjacent normal tissues was also included. All procedures involving human samples were approved by the ethics committee at the First Affiliated Hospital of Xi\u0026rsquo;an Jiaotong University, and written informed consent was obtained from all participants.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunohistochemistry\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFFPE tissue sections (4 \u0026micro;m thick) were mounted and heated at 60\u0026deg;C for 2 hours, and then deparffinized in xylene and rehydrated using an ethanol gradient. Antigen retrieval was conducted by steaming sections in citrate (pH 6.0) or EDTA (pH 8.0) buffers at 95\u0026deg;C for 10 minutes. The activities of endogenous peroxidases were blocked with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 15 minutes. After blocking with 10% BSA (1 hour, ambient temperature), sections were treated overnight with primary antibodies at 4\u0026deg;C. After washing with PBS, the sections were exposed to appropriate secondary antibodies (anti-rabbit or anti-mouse) for 30 minutes, followed by visualization with liquid DAB\u0026thinsp;+\u0026thinsp;substrate (#9018, Golden Bridge, China) and nuclear counterstaining with Carazzi\u0026rsquo;s hematoxylin (#BL702A, Biosharp, China). Two pathologists independently evaluated all stained slides.\u003c/p\u003e\u003cp\u003e\u003cb\u003eqPCR\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotal RNA was extracted from frozen tissue specimens using TRIzol, and homogenization was performed using a mechanical tissue disruptor. RNA was isolated from cells using the RNA-Quick Purification Kit (Yishan Biotechnology, Shanghai, China). A PrimeScript RT reagent Kit (Takara, Japan) was utilized for reverse-transcription of RNA to cDNA, and qPCR was performed with SYBR Green Master Mix (Cwbio, Beijing, China) on an ABI 7500 PCR platform. \u003cem\u003eGAPDH\u003c/em\u003e was the internal reference and relative gene expression was determined using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method. Primer sequences are given in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern blotting and co-immunoprecipitation (co-IP)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWhole-cell lysates were prepared with RIPA buffer (Beyotime, Shanghai, China) with protease and phosphatase inhibitors (Cwbio, Beijing, China) and 1 mM PMSF. For cytoplasmic and nuclear protein fractionation, a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) was utilized as directed. For Co-IP assays, cells were lysed in NP-40 buffer with protease and phosphatase inhibitors and lysates were treated overnight with antibodies against RBBP4, IκBα, or HA at 4\u0026deg;C, followed by capture of the complexes with Protein A /G agarose for 3 hours. Following three washes with PBS-T, elution was performed by boiling in SDS loading buffer and resolution with SDS-PAGE before transfer to PVDF membranes, blocking (5% BSA, 1 hour), and probing with appropriate primary and HRP-conjugated secondary antibodies. Bands were visualized using an enhanced chemiluminescence system (Tanon 5200, Shanghai, China).\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunofluorescence (IF) and multiplex immunofluorescence (mIF)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor single-color IF, cells were grown on coverslips in 6-well plates for 48 hours and fixed with 4% paraformaldehyde. Following PBS washes, cells were permeabilized and blocked with 5% normal goat serum and 0.3% Triton X-100 for 1 hour. Primary antibodies were applied overnight at 4\u0026deg;C. The next day, Alexa Fluor 488- or 555-conjugated donkey anti-rabbit secondary antibodies were added for 1 hour, nuclei were counterstained with DAPI, and cells were imaged with a Leica TCS SP5 confocal microscope (Wetzlar, Germany).\u003c/p\u003e\u003cp\u003emIF staining was conducted using a four-color tyramide signal amplification (TSA) kit (Absin, #abs50012, China), as per the manufacturer\u0026rsquo;s protocol. After antigen retrieval and blocking, tissue sections were treated sequentially with primary antibodies for 30 to 60 minutes at 37\u0026deg;C, followed by HRP-conjugated secondary antibodies and TSA reagents conjugated to Opal dyes. After nuclear staining with DAPI, tissues were mounted in glycerol-gelatin medium for fluorescence imaging.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLive-cell imaging\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo monitor cellular dynamics in real time, cells (3\u0026thinsp;~\u0026thinsp;4\u0026times;10\u003csup\u003e4\u003c/sup\u003e /well) in 0.8 mL of complete medium were inoculated into 24-well plates. Immediately after seeding, time-lapse imaging was initiated using the zenCell Owl live-cell microscopy system (innoME GmbH, Germany) within a standard tissue culture incubator. The system captured one image per hour across 24 parallel channels. Following a 24-hour adhesion period, experimental treatments were applied, and imaging resumed 3 hours post-treatment, continuing until the cultures reached confluency. Image analysis and quantification of cell proliferation were performed using ZenCell Owl software to generate growth curves over time.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMigration and invasion assessments\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor both invasion and migration assays, Transwell inserts (Corning) were employed. In invasion assays, the upper chambers were pre-coated with Matrigel (Solarbio). Cells suspended in serum-free medium at a density of 2.5\u0026times;10⁵ cells per mL were seeded into the upper compartments, with medium supplemented with 20% fetal bovine serum (FBS) added to the lower compartments. Following 24 hours of incubation under standard culture conditions, cells remaining on the upper surface of the membrane were removed, and those that had invaded to the lower surface were fixed using the aforementioned method. The Transwell inserts were then transferred to wells containing diluted DAPI solution (1 \u0026micro;g/mL) for 10 minutes of light-protected incubation, followed by three washes in phosphate-buffered saline (PBS). Representative fields of the cells were imaged using a fluorescence microscope to quantify the invaded cells. Migration assays were performed using an identical protocol, excluding the Matrigel coating step.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA-sequencing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTranscriptomic profiling was carried out on RBBP4-overexpressing cells and vector-transfected controls. After rinsing with cold PBS, cells were trypsinized and lysed in TRIzol for total RNA extraction. RNA library construction and high-throughput sequencing were undertaken on an Illumina HiSeq platform by Applied Protein Technology (Beijing, China). Differentially expressed genes (DEGs) were identified using DESeq2 in R, with the criteria of fold change\u0026thinsp;\u0026gt;\u0026thinsp;1.5 and adjusted \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (see Supplementary Table\u0026nbsp;2). Gene Ontology (GO) and KEGG enrichment analyses were undertaken via the DAVID database, with visual representations of enriched biological processes and pathways created using the Goplot R package.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGlucose and lactate measurements\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCellular glucose uptake was evaluated using a Glucose Oxidase Method Kit (APPLYGEN, China), while lactate generation was quantified using a Lactic Acid Assay Kit (Nanjing Jiancheng Bioengineering Institute, China). For both assays, cells were grown in 6-well plates for 24 hours. Conditioned media were then collected following centrifugation to eliminate cells. The concentration of glucose and lactate in the supernatants was determined as directed.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMetabolic assays\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe metabolic profile of cells, including both the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR), was examined using a Seahorse XF96 Extracellular Flux Analyzer (Agilent Technologies). TNBC cells transfected with RBBP4-targeting siRNA or siNC were inoculated in XF96-well plates at densities of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e /well. Following an 18-hour incubation, the media were replaced with Seahorse XF RPMI (pH 7.4) with 10 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate. Cells were equilibrated in a non-CO₂ incubator for one hour prior to analysis. OCR and ECAR values were recorded in real-time upon sequential injection of metabolic modulators, as per the provided protocol. Following completion of measurements, cells were stained with Hoechst 33342 (Solarbio), imaged using the ImageXpressMicro Confocal system (Molecular Devices), and quantified for normalization purposes. Results were analyzed with Seahorse Wave Pro software (Agilent Technologies).\u003c/p\u003e\u003cp\u003eATP generation was assessed using the Seahorse ATP Rate Assay Kit (Agilent Technologies), involving sequential introduction of oligomycin (1.5 \u0026micro;M) and a rotenone/antimycin A mixture (0.5 \u0026micro;M each). Mitochondrial respiratory function was determined via the Mito Stress Test Kit, using the same concentrations of oligomycin, FCCP (2 \u0026micro;M), and rotenone/antimycin A. Glycolytic capacity was measured with a Glycolytic Rate Assay Kit, following treatment with rotenone/antimycin A, 2-deoxyglucose (2-DG, 50 mM), and vehicle. For glycolytic stress testing, cells were incubated in medium containing only L-glutamine (2 mM) and sequentially treated with D-glucose (10 mM), oligomycin (1 \u0026micro;M), and 2-DG (50 mM). All assays were performed using media containing glucose, glutamine, and pyruvate unless otherwise specified.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo xenograft tumor assays\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTwelve BALB/c nude mice (female, six weeks old) were randomly assigned to two groups. Animals were given subcutaneous injections of 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e HCC1937 cells either stably overexpressing RBBP4 or carrying the control vector, administered into the right hypochondriac region. Tumor formation was monitored beginning one week post-injection, with tumor volumes measured biweekly using the standard formula: \u0026frac12; \u0026times; L \u0026times; W\u0026sup2; (where L\u0026thinsp;=\u0026thinsp;length and W\u0026thinsp;=\u0026thinsp;width). After 24 days, mice were euthanized, and the tumors were collected, imaged, and stained with hematoxylin and eosin (HE) or IHC. All animal experiments were reviewed and approved by the l Animal Care and Use Committee of the Medical College of Xi'an Jiaotong University .\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical analyses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll statistical analyses were conducted using SPSS 19.0 and GraphPad Prism 7. Data are shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation from a minimum of 3 independent biological replicates. For comparison between two groups, an independent samples t-test was used when data met normality assumptions; otherwise, the non-parametric rank sum test was applied. One-way ANOVAs were used for comparisons involving more than two groups. Fisher\u0026rsquo;s exact test was employed for analyzing associations between categorical clinicopathological variables. A \u003cem\u003ep\u003c/em\u003e-value less than 0.05 was deemed significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eRBBP4 upregulation is evident in TNBC and linked to poor prognostic outcomes in patients\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo explore the clinical relevance of RBBP4 in TNBC, its expression was assessed utilizing the UALCAN database. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, RBBP4 transcript levels were significantly elevated in TNBC tissue samples relative to normal tissues (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, survival analysis using the Xiantao database did not reveal a statistically significant difference in patient outcomes based on RBBP4 expression levels (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Given the limitations of the Xiantao dataset as a means of distinguishing TNBC from other breast cancer subtypes, we proceeded with direct analysis of clinical specimens.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, qPCR and IHC approaches were utilized to examine RBBP4 mRNA and protein levels, respectively, in tumor and matched normal samples from 120 TNBC cases who underwent surgical resection between September 2010 and December 2011. Both assays demonstrated a marked upregulation of RBBP4 in tumor tissues relative to adjacent non-tumor tissues (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Kaplan\u0026ndash;Meier curves indicated that high intratumoral RBBP4 levels were linked with reduced overall survival (P\u0026thinsp;=\u0026thinsp;0.0292; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Furthermore, Western blotting of TNBC cells showed that RBBP4 protein levels were markedly raised in HCC1937 cells relative to MDA-MB231 cells (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Collectively, these results indicate that RBBP4 is markedly overexpressed in TNBC and may thus represent a prognostic indicator for this cancer type.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRBBP4 promotes in vitro TNBC cell proliferation without impacting their metastatic potential\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo examine the functions of RBBP4 in TNBC progression, we engineered MDA-MB231 and HCC1937 cell lines to either overexpress or silence RBBP4 using plasmid transfection and siRNA, respectively. Quantitative PCR confirmed significant changes in RBBP4 transcript levels following transfection in both models (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), and these alterations were validated at the protein level via Western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). HCC1937 cells were used for knockdown experiments, while MDA-MB231 cells were employed for overexpression studies. Proliferation was monitored using a zenCELL Owl live-cell imaging platform, capturing hourly images over a 96-hour period. Normalized growth curves and doubling time analyses demonstrated a pronounced proliferative advantage in RBBP4-overexpressing cells compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This finding was further supported by EdU incorporation and colony formation assays, which revealed that RBBP4 knockdown markedly decreased proliferation in HCC1937 cells, while its overexpression enhanced proliferation in MDA-MB231 cells (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo investigate the potential effect of RBBP4 on cellular motility, we performed wound healing and Transwell assays. These experiments revealed no significant alterations in either migration or invasion following RBBP4 overexpression or knockdown (Supplementary Figs. S2 and S3). These results suggest that while RBBP4 facilitates TNBC cell proliferation, it does not modulate metastatic behaviors \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRBBP4 enhances in vivo TNBC tumor growth\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo clarify the effects of RBBP4 on tumor growth in a xenograft model, nude mice were injected with MDA-MB231 cells stably overexpressing RBBP4 or with vector control cells. Biweekly tumor volume measurements indicated that RBBP4-overexpressing cells produced significantly larger tumors over time (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). At the study endpoint, tumors in the RBBP4-overexpressing group exhibited significantly greater weights compared to controls (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Histopathological examination confirmed that no liver metastases were detectable in either group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). IHC analysis of excised tumors showed markedly increased staining for both RBBP4 and the proliferation marker Ki67 in the overexpression group relative to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). These findings confirm that RBBP4 enhances TNBC tumor growth \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eGlycolytic activity driven by RBBP4 facilitates the proliferation of TNBC cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRNA sequencing of RBBP4- overexpressing MDA-MB231 cells identified 93 protein-coding DEGs, including 56 upregulated and 37 downregulated transcripts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA; Supplementary Table\u0026nbsp;2). Functional enrichment analyses using KEGG and GO databases revealed that several pathways involved in metabolic regulation were significantly affected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; Supplementary Figs. S4A, S4B). Notably, the upregulated genes CDC25A and JUN, both of which are implicated in glycolytic activation, were prominently enriched in the RBBP4-overexpressing group. Investigation of glucose uptake and lactate secretion in RBBP4-manipulated cells showed that both parameters were markedly elevated upon RBBP4 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). To further test the dependency of RBBP4-driven proliferation on glycolytic flux, we conducted colony formation assays using 2-DG, a competitive glycolytic inhibitor, and D-galactose, which slows glycolysis due to its delayed entry into the pathway. Both treatments markedly suppressed the pro-proliferative effects induced by RBBP4 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo quantify the metabolic reprogramming more precisely, the ECAR and OCR were determined using Seahorse metabolic analysis. ECAR, an indicator of glycolytic activity, was significantly elevated in RBBP4-overexpressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, G), while OCR, a measure of oxidative phosphorylation, remained unchanged (Supplementary Figs. S5A, S5B). These findings support a model in which RBBP4 enhances glycolytic flux in a manner consistent with the induction of the Warburg effect, thereby promoting TNBC cell proliferation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRBBP4 modulates NF-κB activation and PD-L1 levels\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further explore the downstream molecular pathways regulated by RBBP4, we performed KEGG enrichment analysis and GSEA on transcriptomic data from MDA-MB231 cells with RBBP4 overexpression. These analyses revealed significant upregulation of genes associated with the NF-κB signaling cascade (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Given the established role that NF-κB plays in TNBC pathogenesis, we hypothesized that RBBP4 may enhance tumor progression via activation of this pathway. Among the analyzed members of the NF-κB family, RelB exhibited the most substantial expression change in this experimental context. To determine whether RBBP4 influences RelB activation, we assessed its subcellular localization via nuclear-cytoplasmic fractionation and Western blotting. RBBP4 overexpression promoted nuclear translocation of RelB, accompanied by decreased cytoplasmic retention (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Co-IP assays demonstrated a physical interaction between RBBP4 and RelB (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), and immunofluorescence confirmed their co-localization in the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs NF-κB activation has been linked to PD-L1 upregulation, we next assessed the relationship between RBBP4 and CD274 (PD-L1) levels utilizing information from the Xiantao database[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This showed a marked positive association between RBBP4 and PD-L1 (R\u0026thinsp;=\u0026thinsp;0.156, P\u0026thinsp;=\u0026thinsp;0.002; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Since RelB is a transcription factor capable of interacting with the promoter regions of target genes and has previously been shown to interact with the PD-L1 promoter[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], we conducted chromatin immunoprecipitation (ChIP) followed by PCR to validate this interaction. Our ChIP-PCR results confirmed direct binding between RELB and the PD-L1 promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). To further support this finding, multiplex immunofluorescence analysis of TNBC tissue samples revealed that both RelB and PD-L1 levels were markedly elevated in specimens exhibiting high RBBP4 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). We then employed siRNA-mediated knockdown of RelB to assess its functional relevance. A reduction in RelB expression led to decreased PD-L1 levels, indicating a regulatory relationship. Moreover, colony formation assays indicated that RelB knockdown impaired the clonogenic capacity of TNBC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). These findings thus suggest that RBBP4 raises PD-L1 levels by NF-κB activation through modulation of RelB activity, thereby contributing to TNBC progression.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLactate-induced H3K18la-mediated upregulation of RBBP4 is evident in TNBC\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEmerging evidence suggests that lactate, a metabolic byproduct of glycolysis, can drive protein lactylation, a post-translational modification that influences stability and gene expression. H3K18la lactylation has been the most thoroughly studied form of such modification[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Western blotting showed markedly raised H3K18la levels in TNBC tissues relative to controls, indicating that histone lactylation is a frequent event in TNBC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). To determine whether lactylation regulates RBBP4 expression, we exposed HCC1937 cells to varying lactate concentrations, indicating dose-dependent increases in RBBP4 contents in response to lactate (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Conversely, treatment with 2-DG or oxamate, an LDHA inhibitor, led to a marked suppression of RBBP4 expression in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Notably, oxamate also decreased H3K18la levels, while co-treatment with sodium lactate (Nala) reversed oxamate effects on both RBBP4 and H3K18la level (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). To further examine whether RBBP4 expression is directly regulated by H3K18la, we performed ChIP-qPCR assays in which H3K18la was enriched at the RBBP4 promoter region, with this enrichment having been significantly diminished following treatment with 2-DG or oxamate (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). These findings indicate that lactate produced via glycolytic activity can enhance RBBP4 transcription in TNBC cells through H3K18la-dependent epigenetic regulatory mechanisms.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDysregulated expression of RBBP4 is commonly associated with aberrant chromatin remodeling and disrupted transcriptional[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Our study reveals a multifaceted role for RBBP4 in TNBC, linking its elevated expression to poor clinical outcomes and enhanced tumor aggressiveness. Analysis of the UALCAN data indicated marked upregulation of RBBP4 in TNBC tissues relative to normal counterparts, suggesting its potential utility as a diagnostic biomarker. This initial finding was corroborated by qPCR and immunohistochemistry analyses performed on clinical specimens from 120 TNBC patients, which further demonstrated that elevated RBBP4 levels were associated with significantly reduced survival, positioning RBBP4 as a candidate prognostic biomarker in this cancer type.\u003c/p\u003e\u003cp\u003eUncontrolled proliferation is a hallmark of cancer and is linked to both disease progression and poor clinical outcomes[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Understanding the molecular regulators of cell division is thus essential for identifying therapeutic targets[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In this context, our in vitro assays identified RBBP4 as a promoter of TNBC cell proliferation. Specifically, siRNA-mediated knockdown of RBBP4 in HCC1937 cells led to substantial suppression of proliferation, whereas forced overexpression in MDA-MB231 cells enhanced cell growth. These functional effects were validated through live-cell imaging, EdU incorporation, and colony formation assays. Notably, RBBP4 manipulation had no observable effect on migratory or invasive behaviors in vitro, suggesting that its oncogenic role is primarily confined to promoting cellular proliferation rather than metastasis. To verify the cellular findings, we generated mouse xenograft model using MDA-MB231 cells engineered to overexpress RBBP4. Tumors derived from RBBP4-overexpressing cells exhibited significantly accelerated growth and increased mass relative to control tumors. Histopathological analysis confirmed increased proliferative activity, as indicated by enhanced Ki-67 staining and higher RBBP4 expression. These results, taken together with our in vitro observations, establish RBBP4 as a critical driver of TNBC proliferation, both in culture and in vivo, highlighting its potential in treating the disease.\u003c/p\u003e\u003cp\u003eThe dysregulation of glycolysis is associated with sustaining rapid tumor cell growth and survival under hypoxic conditions[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In addition to its importance as an energy source, glycolysis also facilitates oncogenic signaling and biosynthetic pathways[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Prior studies have identified glycolytic enzymes such as PKM2 and PFKFB3 as oncogenic drivers[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. For instance, PKM2 overexpression is linked with adverse clinical outcomes and transcriptional activation in various cancers[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Similarly, inhibition of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) can lead to the inhibition of angiogenic activity[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Given the centrality of glycolysis to TNBC aggression, RBBP4 likely shapes tumor progression through effects on these mechanisms. NF-κB signaling is also central to progressive tumor growth[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], driving oncogenesis through the activation of genes that favor proliferative, migratory, and invasive activity[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. NF-κB controls glycolytic enzymes at the transcriptional level[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Tumor-associated macrophage (TAM)-derived IL-1α can induce NF-κB activation in TNBC, resulting in enhanced glycolytic activity and cytokine biogenesis[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. RNA-seq analyses and functional enrichment assays unveiled the effects of RBBP4 overexpression on glycolysis and other metabolic pathways, as further evidenced by elevated ECAR levels and higher levels of glucose consumption in these RBBP4-overexpressing cells, triggering the Warburg effect. Such metabolic reprogramming is crucial for the highly proliferative demands of TNBC cells[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The NF-κB pathway was also identified herein as a crucial downstream effector of RBBP4, as its overexpression resulted in NF-κB activation characterized by RelB nuclear translocation and interaction with RBBP4. RelB interaction with the PD-L1 promoter was also noted, consistent with NF-κB-mediated modulation of PD-L1 levels and the consequent ability of tumor cells to establish an immunosuppressive microenvironment. RelB binding to the PD-L1 promoter and consequent PD-L1 upregulation may be indicative of the role of RBBP4 as a modulator of TNBC immune responses through the control of PD-L1 expression, allowing tumors to evade immune-mediated destruction.\u003c/p\u003e\u003cp\u003eAs a glycolysis byproduct, lactate can facilitate lysine lactylation on histones, thereby inducing transcriptional activity[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. H3K18 lactylation and other forms of this epigenetic modification have recently been linked to oncogenic development, progressive tumor growth, metabolic reprogramming, and immune evasion[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In the present study, elevated H3K18 lactylation (H3K18la) levels were noted in TNBC tissue samples, consistent with the prevalence of this modification in this form of cancer. The upregulation of RBBP4 was further noted in response to lactate treatment, whereas it was downregulated in response to the glycolytic inhibitors oxamate and 2DG. Strikingly, the application of the lactate analog Nala resulted in the reversal of oxamate-mediated suppression of RBBP4 and H3K18la levels. ChIP and qPCR results provided further confirmation of H3K18la enrichment at the RBBP4 promoter, with both oxamate and 2DG partially abrogating this enrichment. 2DG and oxamate. Together, these results offer evidence that lactate-mediated H3K18la recruitment to the RBBP4 promoter can lead to the enhanced expression of RBBP4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe study findings highlight RBBP4 as a key regulator of progressive TNBC tumor growth attributable to its ability to modulate metabolic activity, immune functionality, and cellular proliferation. As TNBC tissue samples exhibited elevated RBBP4 levels that were associated with poor patient outcomes, these findings underscore the potential of RBBP4 as a marker and target for the disease. However, future studies exploring the precise mechanisms whereby RBBP4 shapes the progression of TNBC will be essential, as will efforts to validate its performance as a therapeutic target in a clinical context.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003etriple-negative breast cancer (TNBC); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H3K18la, H3 lysine 18 lactylation; IgG, immunoglobulin G; ChIP-qPCR, chromatin immunoprecipitation-quantitative polymerase chain reaction; 2DG, 2-deoxyglucose.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach author approved the manuscript before submission for publication.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe studies involving humans were approved by the ethical committees at the First Affiliated Hospital of Xi\u0026rsquo;an Jiaotong University. The studies were conducted in accordance with the local legislation and institutional requirements, and we have obtained written informed consent from all participants involved in the study. The animal studies were approved by the Medical College of Xi\u0026apos;an Jiaotong University. The studies were conducted in accordance with the local legislation and institutional requirements. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article [and its supplementary information files].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the Natural Science Foundation of Shaanxi Province (2019JM-115, 2018JQ8030, 2025SF-YBXM-385).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXD: Conceptualization, Data curation, Investigation, Methodology, Visualization, Writing \u0026ndash; original draft. HX: Investigation, Methodology, Visualization, Writing \u0026ndash; original draft. PZ: Investigation, Methodology, Visualization, Writing \u0026ndash; original draft. JL: Conceptualization, Investigation, Funding acquisition, Writing \u0026ndash; original draft. SS; Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing \u0026ndash;review \u0026amp; editing. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFilho AM, Laversanne M, Ferlay J, Colombet M, Pieros M, Znaor A, Parkin DM, Soerjomataram I, Bray F. The GLOBOCAN 2022 cancer estimates: Data sources, methods, and a snapshot of the cancer burden worldwide. INTERNATIONAL JOURNAL OF CANCER. 2025;156.\u003c/li\u003e\n\u003cli\u003eLigorio F, Vingiani A, Torelli T, Sposetti C, Drufuca L, Iannelli F, Zanenga L, Depretto C, Folli S, Scaperrotta G, et al. 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Heliyon. 2024;10:e38426.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Triple-negative breast cancer, RBBP4, Glycolysis, NF-κB, PD-L1","lastPublishedDoi":"10.21203/rs.3.rs-7280626/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7280626/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eTo investigate the role of retinoblastoma binding protein 4 (RBBP4) in triple-negative breast cancer (TNBC), an aggressive tumor lacking targeted treatments, and explore its potential as a therapeutic target.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eThe study analyzed RBBP4 expression in TNBC tumors and its association with patient survival. Experimental approaches included assessing the impact of RBBP4 on in vitro cellular proliferation and in vivo tumor growth, as well as invasion and migration. Transcriptomic analyses were performed to examine RBBP4-driven metabolic reprogramming. Molecular interactions between RBBP4 and RelB, and the effects of glycolysis-derived lactate on epigenetic regulation, were also investigated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eRBBP4 was upregulated in TNBC tumors, with higher levels inversely associated with patient survival. RBBP4 promoted in vitro TNBC cellular proliferation and in vivo tumor growth but had no effect on invasion or migration. Transcriptomic analyses revealed RBBP4-driven reprogramming of glycolytic metabolism, characterized by Warburg effect-related phenotypes (elevated glucose consumption, lactate generation, and extracellular acidification). At the molecular level, RBBP4 interacted with RelB, activating NF-κB, which led to nuclear RelB translocation and PD-L1 upregulation. Additionally, glycolysis-derived lactate induced H3K18 lactylation, forming a feedforward epigenetic loop that sustained RBBP4 expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eRBBP4 acts as a nodal regulator linking metabolic reprogramming, NF-κB activation, and immune evasion in TNBC. Targeting RBBP4 or its associated downstream pathways may offer viable strategies for managing TNBC.\u003c/p\u003e","manuscriptTitle":"RBBP4 orchestrates glycolytic reprogramming and NF-κB-mediated immune evasion in triple-negative breast cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-17 14:14:18","doi":"10.21203/rs.3.rs-7280626/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e4bb90a8-34e6-4a77-b6c4-7a4d03bbe466","owner":[],"postedDate":"August 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-26T22:30:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-17 14:14:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7280626","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7280626","identity":"rs-7280626","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}