{"paper_id":"484ec715-7ff8-43de-a6cc-0231d7bceed4","body_text":"The IER5L–AK2 axis drives aggressive behavior 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 Article The IER5L–AK2 axis drives aggressive behavior in triple-negative breast cancer xin zhang, Jiani zhang, Yi Li, Xiaoshan Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8661639/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Breast cancer is the most frequently diagnosed malignancy in women worldwide. Triple-negative breast cancer (TNBC) has particularly poor outcomes, largely due to the lack of effective therapeutic targets. Here, we investigated the expression pattern, functional relevance, and potential molecular mechanisms of Immediate Early Response 5-Like gene (IER5L) in breast cancer by integrating public database analyses, clinical tissue validation, in vitro assays, and in vivo xenograft experiments. IER5L was significantly upregulated in breast cancer tissues, with the highest levels observed in TNBC, and elevated IER5L expression was associated with unfavorable prognosis. Silencing IER5L suppressed proliferation and migration of breast cancer cells, increased apoptosis, and inhibited tumor growth in nude mouse xenograft models. Mechanistically, proteomic profiling identified adenylate kinase 2 (AK2) as a key downstream effector of IER5L. IER5L depletion led to reduced AK2 expression and attenuation of STAT3/mTOR-related signaling. Importantly, rescue experiments demonstrated that ectopic AK2 expression partially reversed the inhibitory effects of IER5L knockdown on cell proliferation and migration. Together, these findings suggest that IER5L contributes to breast cancer progression through an AK2-associated STAT3/mTOR signaling program and support IER5L as a potential prognostic biomarker and therapeutic target, particularly in TNBC. Health sciences/Biomarkers Biological sciences/Cancer Health sciences/Oncology Triple-negative breast cancer IER5L AK2 Metabolic reprogramming STAT3/mTOR signaling Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Breast cancer is the most common malignancy among women worldwide, with its incidence continuing to increase and posing a serious threat to women’s health[ 1 ], [ 2 ]. Although substantial advances have been made in screening strategies and therapeutic approaches, the prognosis of breast cancer remains highly heterogeneous[ 3 ], [ 4 ], [ 5 ]. However, patients with recurrent, metastatic, or triple-negative breast cancer (TNBC) still have a poor prognosis, and these cases remain a major challenge in treatment[ 6 ], [ 7 ], [ 8 ]. Immediate-early response genes (IERGs) are a class of genes that can be rapidly induced following cellular stress stimuli, such as DNA damage, oxidative stress, and metabolic pressure[ 9 ]. Therefore, IERGs play critical roles in signal transduction, cell cycle regulation, cell survival, and tumor progression. Increasing evidence suggests that the dysregulation of IERGs contributes to malignant transformation and cancer progression[ 10 ]. Immediate early response 5-like (IER5L), together with immediate early response 2 (IER2) and immediate early response 5 (IER5), belongs to the IER gene family and shares a high degree of structural homology in the N-terminal region, suggesting potential functional conservation among these members. Previous studies have shown that IER2 is associated with the occurrence of colorectal cancer and hepatocellular carcinoma, and can serve as a biomarker for colorectal cancer, hepatocellular carcinoma, and melanoma[ 11 ], [ 12 ], [ 13 ]. Studies have demonstrated that IER5L can promote prostate cancer cell proliferation and metastasis through the regulation of PP2A activity[ 14 ]. In parallel, IER5 has been shown to participate in cellular stress adaptation through the PP2A/HSF1-associated phosphorylation regulatory network, and its abnormal activation has been linked to ovarian cancer cell proliferation and peritoneal dissemination[ 15 ][ 16 ]. These findings collectively underscore the important roles of IER family members in tumor stress responses and malignant phenotypes. Despite the established role of IER5L, its biological function and underlying molecular mechanisms in breast cancer remain largely unexplored. Against this backdrop, the present study integrates functional experiments and mechanistic analyses to systematically investigate the role of the IER5L–AK2 axis, aiming to elucidate the potential molecular mechanisms by which IER5L contributes to breast cancer development, thereby providing insights into the potential of IER5L as a prognostic biomarker and therapeutic target in breast cancer. Results IER5L is upregulated in breast cancer and is associated with poor prognosis in TNBC To evaluate the potential involvement of IER5L in breast cancer, we first analyzed tumor and normal tissue datasets from GEO and METABRIC. Expression validation using GEPIA3.0 and TIMER3.0 consistently showed that IER5L expression was significantly higher in breast cancer tissues than in normal breast tissues (Fig. 1 A). Kaplan–Meier analyses further indicated that higher IER5L expression was associated with worse overall survival (Fig. 1 B). We next performed multivariate Cox regression analyses and observed that IER5L expression differed across subgroups stratified by age, clinical stage, and histological grade (Fig. 1 C). To facilitate potential clinical translation, we constructed a nomogram incorporating IER5L expression and clinicopathological variables to estimate outcome probabilities (Fig. 1 D). To validate these bioinformatic findings in clinical specimens, we examined IER5L expression in a tissue microarray consisting of 150 TNBC samples and 30 matched adjacent non-tumor tissues. Immunohistochemistry and Mann–Whitney U testing confirmed significantly higher IER5L levels in tumor tissues (Fig. 1 E; Table 1 ). Clinicopathological analyses demonstrated significant associations between IER5L expression and age, pathological grade, and tumor size (Table 2 ). Spearman correlation analyses further showed that IER5L expression was positively correlated with pathological grade and tumor size, and negatively correlated with age (Table 3 ). Collectively, these results indicate that IER5L is frequently overexpressed in breast cancer, particularly TNBC, and that higher IER5L expression is associated with more aggressive clinicopathological features and poorer prognosis. Table 1 The Mann–Whitney U test confirms that IER5L expression is significantly higher in tumor tissues compared to adjacent normal tissues. IER5L expression Tumor tissue Para-carcinoma tissue p value Cases Percentage Cases Percentage P < 0.001 Low 37 26.81% 30 100.0% High 101 73.19% 0 0% Table 2 IER5L expression shows statistically significant associations with different age groups, pathological grades, and tumor sizes. IER5L expression Tumor tissue Para-carcinoma tissue p value Cases Percentage Cases Percentage P < 0.001 Low 37 26.81% 30 100.0% High 101 73.19% 0 0% Table 3 Spearman correlation analysis further reveals that IER5L expression is positively correlated with pathological grade and tumor size, and negatively correlated with patient age. IER5L Age Spearman’s ρ -0.187 P value (two-tailed) 0.028 N 138 Grade Spearman’s ρ 0.304 P value (two-tailed) P<0.001 N 138 Tumor size Spearman’s ρ 0.209 P value (two-tailed) 0.014 N 138 IER5L knockdown suppresses the proliferation and migration of breast cancer cells To determine whether IER5L is expressed in breast cancer cell lines, we measured IER5L mRNA levels using qRT-PCR. IER5L expression was significantly higher in BT-549, MCF-7, and MDA-MB-231 cells than in the non-tumorigenic mammary epithelial cell line MCF-10A (Fig. 2 A). MDA-MB-231 and BT-549 cells exhibited the highest expression and were selected for subsequent functional analyses. We established stable IER5L knockdown models using lentiviral shRNAs. qRT-PCR confirmed efficient silencing, with shIER5L-2 and shIER5L-3 achieving the greatest knockdown (MDA-MB-231: 85.3% and 80.1%; BT-549: 95.2% and 90.7%, respectively) (Fig. 2 B). Western blotting further verified reduced IER5L protein abundance in all knockdown groups compared with controls (Fig. 2 C). Functionally, CCK-8 assays showed that IER5L knockdown significantly reduced cell viability over time (Fig. 2 D), and colony formation assays confirmed impaired long-term proliferative capacity (Fig. 2 E). Flow cytometric analysis revealed a marked increase in apoptosis in IER5L-silenced cells (Fig. 2 F). In addition, Transwell assays demonstrated significantly reduced migration following IER5L knockdown (Fig. 2 G). These results indicate that IER5L supports proliferative and migratory phenotypes in breast cancer cells. IER5L knockdown inhibits xenograft tumor growth in vivo To assess the in vivo relevance of IER5L, we used a nude mouse xenograft model. MDA-MB-231 cells stably expressing shIER5L or control shRNA were implanted subcutaneously. Tumor growth was significantly slower in the shIER5L group, and tumor weights were reduced at endpoint (Fig. 2 H–J). In vivo fluorescence imaging also showed reduced signal intensity in the shIER5L group throughout the experiment (Fig. 2 K). Consistent with these observations, western blot analyses of xenograft tissues confirmed reduced IER5L protein levels in the knockdown group (Fig. 2 L). Immunohistochemistry further showed decreased IER5L staining intensity and lower Ki-67 expression in tumors derived from IER5L-silenced cells (Fig. 2 M, N), indicating reduced proliferative activity. Together, these in vivo results support an important role of IER5L in breast cancer tumor growth. AK2 is a key downstream mediator associated with IER5L-dependent signaling To explore pathways potentially influenced by IER5L, we performed a comparative protein kinase array in IER5L-overexpressing cells and control cells. IER5L overexpression was accompanied by increased phosphorylation of multiple signaling proteins, including STAT3, YES, and PRAS40 (Fig. 3 A), suggesting that IER5L may be linked to activation of oncogenic signaling cascades. We then assessed these candidates in IER5L knockdown models. Western blot analyses showed that IER5L silencing reduced phosphorylation of STAT3, YES, and PRAS40, whereas total protein levels were largely unchanged (Fig. 3 B). These findings suggest that IER5L is more closely associated with signaling activation status than with baseline protein abundance and point to a relationship with STAT3/mTOR-related pathways. To identify downstream effectors, we performed iTRAQ-based quantitative proteomic profiling in IER5L-silenced cells and integrated these results with TCGA analyses. Among proteins significantly downregulated following IER5L knockdown, CNN3, AK2, and HMGCS1 were highly expressed in TNBC and associated with poorer outcomes. Notably, AK2 exhibited the most pronounced decrease (Fig. 3 C). Western blot validation confirmed substantial reduction of AK2 protein levels upon IER5L knockdown, whereas CNN3 and HMGCS1 showed more modest decreases (Fig. 3 D). To test the functional relevance of AK2, we performed rescue experiments by overexpressing AK2 in IER5L-silenced MDA-MB-231 and BT-549 cells. Western blotting confirmed efficient IER5L knockdown and robust AK2 overexpression (Fig. 3 E). Functionally, IER5L silencing significantly reduced proliferation (CCK-8) and migration (Transwell), and ectopic AK2 expression partially restored both phenotypes (Fig. 3 F, G). These results indicate that AK2 contributes to IER5L-associated malignant behaviors, although the partial nature of the rescue suggests additional downstream pathways may also be involved. Discussion In this study, we systematically investigated the biological role of IER5L in breast cancer and identified an IER5L–AK2–associated regulatory axis that links metabolic regulation with malignant phenotypes. By integrating bioinformatic analyses, clinical tissue validation, in vitro and in vivo functional assays, and phosphoproteomic profiling, we provide multi-level evidence supporting the involvement of IER5L in breast cancer progression. We first evaluated the expression pattern and clinical relevance of IER5L. Our analyses demonstrated that IER5L is significantly upregulated in breast cancer tissues compared with normal breast tissues, and that higher IER5L expression is associated with poorer overall survival. Multivariate Cox regression analysis further indicated that IER5L expression serves as an independent prognostic factor. In addition, elevated IER5L levels were associated with younger patient age (< 60 years), more advanced clinical stage (II–IV), and higher histological grade (grade 3). These associations suggest that IER5L expression may increase with tumor aggressiveness and may be involved in both tumor development and progression. IER5L is a member of the Immediate Early Response Genes (IERGs) family, which are rapidly induced in response to various cellular stress conditions, including DNA damage, chemotherapy exposure, and metabolic stress[ 17 ], [ 18 ], [ 19 ]. Consistent with this functional background, our experimental data showed that IER5L knockdown significantly inhibited breast cancer cell proliferation and migration, promoted apoptosis, and suppressed tumor growth in xenograft models. These findings indicate that IER5L contributes to tumor cell survival and growth, potentially conferring an adaptive advantage under stress conditions commonly encountered in the tumor microenvironment. At the mechanistic level, we identified AK2 as an important downstream effector associated with IER5L-mediated regulation. AK2 is a mitochondrial intermembrane enzyme that plays a central role in cellular energy homeostasis by catalyzing the interconversion of ATP and AMP. Previous studies have shown that elevated lactylation of AK2 is associated with unfavorable prognosis in hepatocellular carcinoma[ 20 ], [ 21 ], [ 22 ]. Silencing IER5L led to a marked reduction in AK2 expression, and ectopic AK2 overexpression partially restored the proliferative capacity of IER5L-silenced cells. These results support a functional link between IER5L and AK2, although they do not establish direct regulation. Instead, the data suggest that AK2 acts as a downstream component within a broader IER5L-associated regulatory network. Beyond AK2, protein kinase array analysis and subsequent Western blot validation revealed that IER5L influences the phosphorylation status of multiple signaling molecules, including STAT3, PRAS40, and YES, without substantially altering their total protein levels. These signaling components are closely connected to the JAK/STAT and mTOR pathways, which are known to integrate oncogenic signaling with metabolic control[ 23 ], [ 24 ], [ 25 ]. Together, these observations suggest that IER5L may function as an upstream regulatory node that coordinates metabolic regulation and proliferative signaling through multiple interconnected pathways. Based on these findings, we propose a working model (Fig. 4 ) in which elevated IER5L expression is associated with enhanced mitochondrial energy metabolism via AK2 and increased activation of STAT3/mTOR-related signaling. Through this coordinated regulation, IER5L may contribute to metabolic reprogramming and support proliferative and migratory phenotypes in breast cancer cells. Importantly, this model represents a proposed regulatory framework derived from the experimental evidence presented and does not imply direct molecular interactions. Several limitations of the present study should be acknowledged. First, although our data demonstrate a functional association between IER5L and AK2, the precise molecular mechanisms underlying this relationship remain unclear and may involve intermediate regulators. Second, the potential impact of IER5L on the tumor immune microenvironment and on therapeutic resistance was not addressed and warrants further investigation. Third, while our analyses focused primarily on triple-negative breast cancer, whether the IER5L–AK2 axis operates similarly across other breast cancer subtypes requires validation in larger and more diverse patient cohorts. Future studies should therefore aim to (1) dissect the molecular mechanisms by which the IER5L–AK2 axis influences cellular energy metabolism, and (2) explore how IER5L-driven metabolic alterations affect immune responses and sensitivity to anticancer therapies. From a clinical perspective, our findings suggest that IER5L may serve as a prognostic biomarker and highlight the IER5L–AK2 axis as a potential target for combination therapeutic strategies in breast cancer. Conclusion Our study indicates that IER5L is upregulated in breast cancer, especially TNBC, and is associated with poor prognosis. Functional and mechanistic analyses suggest that IER5L contributes to breast cancer progression through an AK2-associated signaling axis linked to STAT3/mTOR activation. These findings support IER5L as a potential prognostic biomarker and therapeutic target. Methods and Materials Data sources and bioinformatic analyses Gene expression profiles of breast cancer and normal breast tissues were obtained from the GEO database (GSE54002) and analyzed using R. Data were normalized, and differential expression was assessed using standard pipelines. Prognostic analyses were performed using the METABRIC cohort (downloaded via cBioPortal), including clinical variables such as age, stage, and histological grade. Expression and survival associations were further validated using the GEPIA3.0 and TIMER3.0 online platforms. Cell culture and lentiviral transduction MCF-10A human mammary epithelial cells and breast cancer cell lines (MDA-MB-231, MCF-7, BT-549) were purchased from the cell bank of Shanghai Mingjin Biotechnology Co., Ltd., and cultured according to the supplier’s instructions. Lentiviral shRNAs targeting IER5L were used to generate stable knockdown cell lines. Cells transduced with non-targeting shRNA served as controls. For rescue experiments, AK2 was ectopically overexpressed in IER5L-silenced cells using lentiviral constructs. Quantitative real-time PCR (qRT-PCR) Total RNA was isolated using TRIzol. RNA concentration and purity were assessed using a NanoDrop spectrophotometer. cDNA was synthesized from 1 µg of total RNA using Hiscript QRT Supermix for qPCR (gDNA WIPER). qRT-PCR was performed using SYBR Green mastermix (Vazyme Q111-02) on an ABI 7900 system. Melting curve analysis was used to confirm amplification specificity. GAPDH served as an internal reference. Western blotting Cells were lysed in RIPA buffer containing PMSF. Protein samples were denatured in loading buffer, separated by SDS–PAGE, and transferred to PVDF membranes. Membranes were blocked in 5% non-fat milk in TBST and incubated with primary antibodies at 4°C overnight (IER5L 1:1000; β-actin 1:4000). After washing, membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Signals were visualized using ECL substrate and quantified with β-actin or GAPDH as loading controls. Immunohistochemistry (IHC) and H&E staining Paraffin sections were deparaffinized, rehydrated, and subjected to antigen retrieval using citrate or EDTA buffer. Endogenous peroxidase was blocked with 3% H₂O₂, and non-specific binding was blocked with goat serum. Sections were incubated with primary antibodies, followed by secondary antibody incubation and DAB development. Slides were counterstained with hematoxylin, dehydrated, and mounted. H&E staining followed standard procedures. Cell proliferation, colony formation, apoptosis, and migration assays For CCK-8 assays, equal numbers of cells were seeded in 96-well plates and assessed daily by measuring absorbance at 450 nm. Colony formation assays were performed by seeding 500–1000 cells per well in 6-well plates and culturing for 10–14 days, followed by fixation and crystal violet staining. Apoptosis was evaluated using Annexin V-FITC/PI staining and flow cytometry. Migration was assessed using Transwell chambers without Matrigel; migrated cells were fixed, stained, and quantified after 24 h. Xenograft mouse model and in vivo imaging BALB/c nude mice (4–6 weeks old) were purchased from Shanghai Yaokang Biotechnology Co., Ltd. and used for xenograft experiments. Mice were allocated to experimental groups according to the study design, with an average body weight of 17.75 g in the experimental group and 17.85 g in the control group at the beginning of the experiments. Mice were subcutaneously injected with MDA-MB-231 cells expressing control shRNA or shIER5L. Tumor growth was monitored at predefined time points, and in vivo fluorescence imaging was performed to assess tumor burden. Tumors were excised, weighed, and processed for western blotting and immunohistochemistry (IHC). All procedures were performed under inhalational anesthesia using isoflurane, and humane endpoints were applied to minimize animal suffering. At the end of the experiments, mice were euthanized by CO₂ inhalation followed by cervical dislocation in accordance with institutional guidelines. Tissue microarray cohort and ethics TNBC tissue microarrays (150 TNBC and 30 adjacent tissues) were purchased from Shanghai Mijian Biotechnology Co., Ltd. Ethical approval was provided by the supplier’s ethics committee (Ethics Approval No.: SHYJS-CP-2210009). Proteomic analysis and candidate selection Proteins downregulated after IER5L knockdown were identified by iTRAQ-based proteomic profiling and cross-referenced with TCGA analyses for expression and prognostic relevance in TNBC. Candidate proteins were selected using the following criteria: differential protein logFC < − 0.6; breast cancer expression log2FoldChange.exp > 1; HR.val.optimal.os > 1; and P.val.optimal.os < 0.05. CNN3, AK2, and HMGCS1 were prioritized for validation. Statistical analysis Data analyses were performed using RStudio and SPSS 27. Graphs were generated using Prism 9. Unless otherwise stated, data are presented as mean ± SD from at least three independent experiments. Two-group comparisons were performed using appropriate parametric or non-parametric tests. A two-sided P value < 0.05 was considered statistically significant. Declarations Animal reporting and ethical approval All animal experiments were conducted in accordance with institutional guidelines and approved by the Animal Ethics Committee of Sichuan Provincial People’s Hospital (Approval No.AF/SW-05/01.1). This study is reported in compliance with the ARRIVE guidelines. BALB/c nude mice (4–6 weeks old) were used for xenograft experiments and were allocated to experimental groups according to the study design. Tumor growth was monitored at predefined time points, and humane endpoints were applied to minimize suffering. At the end of the experiments, mice were euthanized by CO₂ inhalation followed by cervical dislocation in accordance with institutional guidelines. Ethics approval and consent to participate Human tissue microarray samples (150 triple-negative breast cancer tissues and 30 adjacent non-tumor tissues) were purchased from Shanghai Xinchao Biotechnology Co.Ltd. Ethical approval for the use of these samples was obtained from the supplier’s ethics committee (Ethics Approval No.SHYJS-CP-2210009). All samples were provided in a de-identified form. The study was conducted in accordance with the Declaration of Helsinki and relevant institutional guidelines and regulations. Informed consent was obtained by the supplier from the participants or was waived by the ethics committee in accordance with applicable regulations. Competing Interests The authors declare that they have no competing interests. The corresponding authors confirm that they are responsible for submitting this declaration on behalf of all authors. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author Contribution Y.L. and X.W. conceived and supervised the study.X.Z. performed the experiments and drafted the manuscript.J.Z. assisted with the experimental work.All authors reviewed and approved the final manuscript. Acknowledgements The authors thank all patients who contributed tissue samples to this study. We acknowledge the technical support provided by the experimental platform staff at the School of Medicine, University of Electronic Science and Technology of China, and the assistance of the animal facility staff during the in vivo experiments. We also thank the contributors of the GEO, METABRIC, TCGA, GEPIA, and TIMER databases for making their datasets publicly available. Data Availability Publicly available datasets were used in this study, including GEO (GSE54002) and METABRIC (accessed via cBioPortal). Analyses were also performed using the GEPIA3.0 and TIMER3.0 online platforms. The datasets generated and/or analyzed during the current study, including raw and processed data supporting the findings, are available from the corresponding author upon reasonable request. References F. Bray et al. , “Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries,” CA A Cancer J Clinicians , vol. 74, no. 3, pp. 229–263, May 2024, doi: 10.3322/caac.21834. R. L. Siegel, T. B. Kratzer, A. N. Giaquinto, H. Sung, and A. Jemal, “Cancer statistics, 2025,” CA A Cancer J Clinicians , vol. 75, no. 1, pp. 10–45, Jan. 2025, doi: 10.3322/caac.21871. X. Xiong et al. , “Breast cancer: pathogenesis and treatments,” Sig Transduct Target Ther , vol. 10, no. 1, p. 49, Feb. 2025, doi: 10.1038/s41392-024-02108-4. H. 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Liao et al. , “Targeting the Warburg effect: A revisited perspective from molecular mechanisms to traditional and innovative therapeutic strategies in cancer,” Acta Pharmaceutica Sinica B , vol. 14, no. 3, pp. 953–1008, Mar. 2024, doi: 10.1016/j.apsb.2023.12.003. Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 16 May, 2026 Reviewers agreed at journal 14 May, 2026 Reviewers invited by journal 06 Feb, 2026 Editor assigned by journal 06 Feb, 2026 Editor invited by journal 05 Feb, 2026 Submission checks completed at journal 31 Jan, 2026 First submitted to journal 31 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-8661639\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":587544751,\"identity\":\"42c1635e-ea8e-41e3-81ac-6bc02b4905d8\",\"order_by\":0,\"name\":\"xin zhang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Electronic Science and Technology of China\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"xin\",\"middleName\":\"\",\"lastName\":\"zhang\",\"suffix\":\"\"},{\"id\":587544752,\"identity\":\"d56697a3-7e4c-4d1e-ab9a-8990192d6c3a\",\"order_by\":1,\"name\":\"Jiani zhang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Electronic Science and Technology of China\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jiani\",\"middleName\":\"\",\"lastName\":\"zhang\",\"suffix\":\"\"},{\"id\":587544753,\"identity\":\"54757eec-6f38-4186-ad10-bd8fea867c26\",\"order_by\":2,\"name\":\"Yi Li\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYBACPmYGhgMMDDZAZgJc0ACvFjaIljSYFgMitECow6RoYecxPFzZdj6anz2BTeLDnz/yDOzN2yQYau7gcRiPwcEzZ27nzux5wCY5s83AsIHnWJkEw7Fn+LU0VNzO3XAjgU2at8EggUEix0yCseEwAS0G53L3g7T8+QPUIv+GGC0VB3I3SAC1MLCBbOEhpIWt4GDDmeTcGWceNlv2thkbtvGkFVskHMOthZ//8OaPjW12uf3tyQdv/PgjJ8/PfnjjjQ81uLUwMHDAYoGxAWIviEjAo4GBgf0BXulRMApGwSgYBQwAkO1OjnmK+wUAAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Yi\",\"middleName\":\"\",\"lastName\":\"Li\",\"suffix\":\"\"},{\"id\":587544754,\"identity\":\"d0ba7c38-b5a7-4dea-9447-369733ea5f7b\",\"order_by\":3,\"name\":\"Xiaoshan Wang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xiaoshan\",\"middleName\":\"\",\"lastName\":\"Wang\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2026-01-21 15:39:37\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-8661639/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-8661639/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":102745606,\"identity\":\"b9034cc7-3566-4857-85bf-40a9f44d94e9\",\"added_by\":\"auto\",\"created_at\":\"2026-02-16 08:52:42\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":940115,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eIER5L is upregulated in breast cancer and is associated with clinical outcome.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Analysis of the GEPIA3.0 database shows that IER5L expression is higher in breast cancer tissues than in normal breast tissues.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) Kaplan–Meier survival analysis using the TIMER3.0 database indicates that higher IER5L expression is associated with poorer overall survival (OS), progression-free survival (PFS), and disease-specific survival (DSS) (P \\u0026lt; 0.05).\\u003c/p\\u003e\\n\\u003cp\\u003e(C) Elevated IER5L expression is associated with clinicopathological features, including age, tumor stage, and histological grade.\\u003c/p\\u003e\\n\\u003cp\\u003e(D) A nomogram incorporating IER5L expression and clinical variables was constructed to estimate survival probabilities at different time points.\\u003c/p\\u003e\\n\\u003cp\\u003e(E) H＆E staining and immunohistochemistry showed that IER5L expression was significantly higher in tumor tissues than in adjacent normal tissues.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8661639/v1/8a5dce4aa56769a699cf2bb5.png\"},{\"id\":102434067,\"identity\":\"0319a6db-0e14-498c-bb33-f40b6f3e4374\",\"added_by\":\"auto\",\"created_at\":\"2026-02-11 15:50:05\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1891530,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eKnocking down IER5L can suppress the proliferation, migration and promote apoptosis of breast cancer cells in vitro and inhibit tumor growth in vivo.\\u003c/strong\\u003e\\u003cbr\\u003e\\n(A) The expression level of IER5L in multiple breast cancer cell lines (BT-549, MCF-7, and MDA-MB-231) was significantly higher than that in normal breast epithelial cell line ( MCF-10A).\\u003cbr\\u003e\\n(B) qPCR analysis showing the knockdown efficiency of IER5L in MDA-MB-231 and BT-549 cells, with shIER5L-2 and shIER5L-3 exhibiting higher silencing efficiency.\\u003cbr\\u003e\\n(C) Western blot analysis confirming reduced IER5L protein expression in IER5L-knockdown cells compared with control cells.\\u003cbr\\u003e\\n(D) CCK-8 assays showing reduced cell viability following IER5L knockdown in MDA-MB-231 and BT-549 cells.\\u003cbr\\u003e\\n(E) Colony formation assays showing decreased long-term proliferative capacity in IER5L-knockdown cells.\\u003cbr\\u003e\\n(F) Flow cytometric analysis indicating increased apoptosis in IER5L-silenced cells.\\u003cbr\\u003e\\n(G) Transwell migration assays showing reduced migratory capacity after IER5L knockdown.\\u003cbr\\u003e\\n(H–J) IER5L knockdown significantly inhibited xenograft tumor growth, resulting in decreased tumor volume and weight, without affecting mouse body weight.\\u003c/p\\u003e\\n\\u003cp\\u003e(K) In vivo fluorescence imaging showing reduced tumor-associated fluorescence intensity in the IER5L-knockdown group.\\u003cbr\\u003e\\n(L) Western blot analysis of xenograft tumor tissues confirming decreased IER5L protein levels in the knockdown group.\\u003cbr\\u003e\\n(M–N) Immunohistochemical showing reduced Ki-67 and IER5L expression in tumors derived from IER5L-knockdown cells.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8661639/v1/eee307205773080f57860205.png\"},{\"id\":102434066,\"identity\":\"a7431819-03a7-4dcb-bc92-6c08cd09bc2f\",\"added_by\":\"auto\",\"created_at\":\"2026-02-11 15:50:05\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1469105,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eAK2 is associated with IER5L-mediated regulation of breast cancer cell proliferation and migration.\\u003c/strong\\u003e\\u003cbr\\u003e\\n(A) Protein kinase array analysis showing increased phosphorylation of multiple signaling proteins, including STAT3, YES, and PRAS40, in IER5L-overexpressing cells compared with control cells.\\u003cbr\\u003e\\n(B) Western blot analysis showing reduced phosphorylation levels of STAT3, YES, and PRAS40 in MDA-MB-231 and BT-549 cells following IER5L knockdown, while total protein levels remain largely unchanged.\\u003cbr\\u003e\\n(C) Integrated iTRAQ-based quantitative proteomic profiling and TCGA analyses identifying candidate downstream proteins downregulated after IER5L knockdown, including CNN3, AK2, and HMGCS1.\\u003cbr\\u003e\\n(D) Western blot validation showing reduced AK2 protein expression after IER5L knockdown, with more modest changes observed for CNN3 and HMGCS1.\\u003cbr\\u003e\\n(E) Western blot analysis confirming efficient IER5L knockdown and AK2 overexpression in MDA-MB-231 and BT-549 cells used for rescue experiments.\\u003cbr\\u003e\\n(F) CCK-8 assays showing that AK2 overexpression partially restores cell viability in IER5L-silenced cells.\\u003cbr\\u003e\\n(G) Transwell migration assays showing partial recovery of migratory capacity following AK2 overexpression in IER5L-knockdown cells.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8661639/v1/c68f3b6fc79e7f6f68ed0d8e.png\"},{\"id\":102434065,\"identity\":\"4a62ad23-f261-4169-b726-9686924c0146\",\"added_by\":\"auto\",\"created_at\":\"2026-02-11 15:50:05\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":195661,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eProposed schematic model illustrating the role of the IER5L–AK2 axis in breast cancer progression.\\u003c/strong\\u003eThis schematic summarizes the main findings of the present study. Elevated IER5L expression is associated with increased AK2 levels and enhanced activation of STAT3/mTOR-related signaling pathways. Through these signaling events, IER5L may contribute to metabolic reprogramming and support proliferative and migratory phenotypes in breast cancer cells. This model is based on the experimental evidence presented in this study and represents a proposed regulatory framework rather than direct molecular interactions.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8661639/v1/862362d210d50b7073dac863.png\"},{\"id\":102750223,\"identity\":\"6a67ebf7-35e5-4cec-929b-78db91ded72d\",\"added_by\":\"auto\",\"created_at\":\"2026-02-16 09:18:35\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":5266490,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8661639/v1/24bbaa7e-bf68-43c8-83ce-7c68613c5a79.pdf\"},{\"id\":102434068,\"identity\":\"18f7bbbe-3ca8-41da-91f0-39ce53ed3743\",\"added_by\":\"auto\",\"created_at\":\"2026-02-11 15:50:06\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":7548300,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryInformation.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8661639/v1/656c042dbf64310c70104b03.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"\\u003cp\\u003eThe IER5L–AK2 axis drives aggressive behavior in triple-negative breast cancer\\u003c/p\\u003e\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eBreast cancer is the most common malignancy among women worldwide, with its incidence continuing to increase and posing a serious threat to women\\u0026rsquo;s health[\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. Although substantial advances have been made in screening strategies and therapeutic approaches, the prognosis of breast cancer remains highly heterogeneous[\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. However, patients with recurrent, metastatic, or triple-negative breast cancer (TNBC) still have a poor prognosis, and these cases remain a major challenge in treatment[\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eImmediate-early response genes (IERGs) are a class of genes that can be rapidly induced following cellular stress stimuli, such as DNA damage, oxidative stress, and metabolic pressure[\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. Therefore, IERGs play critical roles in signal transduction, cell cycle regulation, cell survival, and tumor progression. Increasing evidence suggests that the dysregulation of IERGs contributes to malignant transformation and cancer progression[\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eImmediate early response 5-like (IER5L), together with immediate early response 2 (IER2) and immediate early response 5 (IER5), belongs to the IER gene family and shares a high degree of structural homology in the N-terminal region, suggesting potential functional conservation among these members. Previous studies have shown that IER2 is associated with the occurrence of colorectal cancer and hepatocellular carcinoma, and can serve as a biomarker for colorectal cancer, hepatocellular carcinoma, and melanoma[\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. Studies have demonstrated that IER5L can promote prostate cancer cell proliferation and metastasis through the regulation of PP2A activity[\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. In parallel, IER5 has been shown to participate in cellular stress adaptation through the PP2A/HSF1-associated phosphorylation regulatory network, and its abnormal activation has been linked to ovarian cancer cell proliferation and peritoneal dissemination[\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e][\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. These findings collectively underscore the important roles of IER family members in tumor stress responses and malignant phenotypes.\\u003c/p\\u003e \\u003cp\\u003eDespite the established role of IER5L, its biological function and underlying molecular mechanisms in breast cancer remain largely unexplored. Against this backdrop, the present study integrates functional experiments and mechanistic analyses to systematically investigate the role of the IER5L\\u0026ndash;AK2 axis, aiming to elucidate the potential molecular mechanisms by which IER5L contributes to breast cancer development, thereby providing insights into the potential of IER5L as a prognostic biomarker and therapeutic target in breast cancer.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eIER5L is upregulated in breast cancer and is associated with poor prognosis in TNBC\\u003c/h2\\u003e \\u003cp\\u003eTo evaluate the potential involvement of IER5L in breast cancer, we first analyzed tumor and normal tissue datasets from GEO and METABRIC. Expression validation using GEPIA3.0 and TIMER3.0 consistently showed that IER5L expression was significantly higher in breast cancer tissues than in normal breast tissues (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA). Kaplan\\u0026ndash;Meier analyses further indicated that higher IER5L expression was associated with worse overall survival (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eWe next performed multivariate Cox regression analyses and observed that IER5L expression differed across subgroups stratified by age, clinical stage, and histological grade (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC). To facilitate potential clinical translation, we constructed a nomogram incorporating IER5L expression and clinicopathological variables to estimate outcome probabilities (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD).\\u003c/p\\u003e \\u003cp\\u003eTo validate these bioinformatic findings in clinical specimens, we examined IER5L expression in a tissue microarray consisting of 150 TNBC samples and 30 matched adjacent non-tumor tissues. Immunohistochemistry and Mann\\u0026ndash;Whitney U testing confirmed significantly higher IER5L levels in tumor tissues (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eE; Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). Clinicopathological analyses demonstrated significant associations between IER5L expression and age, pathological grade, and tumor size (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). Spearman correlation analyses further showed that IER5L expression was positively correlated with pathological grade and tumor size, and negatively correlated with age (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). Collectively, these results indicate that IER5L is frequently overexpressed in breast cancer, particularly TNBC, and that higher IER5L expression is associated with more aggressive clinicopathological features and poorer prognosis.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eThe Mann\\u0026ndash;Whitney U test confirms that IER5L expression is significantly higher in tumor tissues compared to adjacent normal tissues.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"6\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003eIER5L expression\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c3\\\" namest=\\\"c2\\\"\\u003e \\u003cp\\u003eTumor tissue\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e \\u003cp\\u003ePara-carcinoma tissue\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003ep value\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCases\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003ePercentage\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eCases\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003ePercentage\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\" morerows=\\\"2\\\" rowspan=\\\"3\\\"\\u003e \\u003cp\\u003eP\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eLow\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e37\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e26.81%\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e30\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e100.0%\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eHigh\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e101\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e73.19%\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0%\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eIER5L expression shows statistically significant associations with different age groups, pathological grades, and tumor sizes.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"6\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003eIER5L expression\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c3\\\" namest=\\\"c2\\\"\\u003e \\u003cp\\u003eTumor tissue\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e \\u003cp\\u003ePara-carcinoma tissue\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003ep value\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCases\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003ePercentage\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eCases\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003ePercentage\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\" morerows=\\\"2\\\" rowspan=\\\"3\\\"\\u003e \\u003cp\\u003eP\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eLow\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e37\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e26.81%\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e30\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e100.0%\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eHigh\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e101\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e73.19%\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0%\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab3\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 3\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eSpearman correlation analysis further reveals that IER5L expression is positively correlated with pathological grade and tumor size, and negatively correlated with patient age.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"3\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eIER5L\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eAge\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eSpearman\\u0026rsquo;s ρ\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e-0.187\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eP value (two-tailed)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.028\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eN\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e138\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eGrade\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eSpearman\\u0026rsquo;s ρ\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.304\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eP value (two-tailed)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eP\\u0026lt;0.001\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eN\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e138\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTumor size\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eSpearman\\u0026rsquo;s ρ\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.209\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eP value (two-tailed)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.014\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eN\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e138\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eIER5L knockdown suppresses the proliferation and migration of breast cancer cells\\u003c/h3\\u003e\\n\\u003cp\\u003eTo determine whether IER5L is expressed in breast cancer cell lines, we measured IER5L mRNA levels using qRT-PCR. IER5L expression was significantly higher in BT-549, MCF-7, and MDA-MB-231 cells than in the non-tumorigenic mammary epithelial cell line MCF-10A (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA). MDA-MB-231 and BT-549 cells exhibited the highest expression and were selected for subsequent functional analyses.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eWe established stable IER5L knockdown models using lentiviral shRNAs. qRT-PCR confirmed efficient silencing, with shIER5L-2 and shIER5L-3 achieving the greatest knockdown (MDA-MB-231: 85.3% and 80.1%; BT-549: 95.2% and 90.7%, respectively) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). Western blotting further verified reduced IER5L protein abundance in all knockdown groups compared with controls (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC).\\u003c/p\\u003e \\u003cp\\u003eFunctionally, CCK-8 assays showed that IER5L knockdown significantly reduced cell viability over time (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD), and colony formation assays confirmed impaired long-term proliferative capacity (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eE). Flow cytometric analysis revealed a marked increase in apoptosis in IER5L-silenced cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eF). In addition, Transwell assays demonstrated significantly reduced migration following IER5L knockdown (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eG). These results indicate that IER5L supports proliferative and migratory phenotypes in breast cancer cells.\\u003c/p\\u003e\\n\\u003ch3\\u003eIER5L knockdown inhibits xenograft tumor growth in vivo\\u003c/h3\\u003e\\n\\u003cp\\u003eTo assess the in vivo relevance of IER5L, we used a nude mouse xenograft model. MDA-MB-231 cells stably expressing shIER5L or control shRNA were implanted subcutaneously. Tumor growth was significantly slower in the shIER5L group, and tumor weights were reduced at endpoint (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eH\\u0026ndash;J). In vivo fluorescence imaging also showed reduced signal intensity in the shIER5L group throughout the experiment (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eK).\\u003c/p\\u003e \\u003cp\\u003eConsistent with these observations, western blot analyses of xenograft tissues confirmed reduced IER5L protein levels in the knockdown group (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eL). Immunohistochemistry further showed decreased IER5L staining intensity and lower Ki-67 expression in tumors derived from IER5L-silenced cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eM, N), indicating reduced proliferative activity. Together, these in vivo results support an important role of IER5L in breast cancer tumor growth.\\u003c/p\\u003e\\n\\u003ch3\\u003eAK2 is a key downstream mediator associated with IER5L-dependent signaling\\u003c/h3\\u003e\\n\\u003cp\\u003eTo explore pathways potentially influenced by IER5L, we performed a comparative protein kinase array in IER5L-overexpressing cells and control cells. IER5L overexpression was accompanied by increased phosphorylation of multiple signaling proteins, including STAT3, YES, and PRAS40 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA), suggesting that IER5L may be linked to activation of oncogenic signaling cascades.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eWe then assessed these candidates in IER5L knockdown models. Western blot analyses showed that IER5L silencing reduced phosphorylation of STAT3, YES, and PRAS40, whereas total protein levels were largely unchanged (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB). These findings suggest that IER5L is more closely associated with signaling activation status than with baseline protein abundance and point to a relationship with STAT3/mTOR-related pathways.\\u003c/p\\u003e \\u003cp\\u003eTo identify downstream effectors, we performed iTRAQ-based quantitative proteomic profiling in IER5L-silenced cells and integrated these results with TCGA analyses. Among proteins significantly downregulated following IER5L knockdown, CNN3, AK2, and HMGCS1 were highly expressed in TNBC and associated with poorer outcomes. Notably, AK2 exhibited the most pronounced decrease (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC). Western blot validation confirmed substantial reduction of AK2 protein levels upon IER5L knockdown, whereas CNN3 and HMGCS1 showed more modest decreases (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD).\\u003c/p\\u003e \\u003cp\\u003eTo test the functional relevance of AK2, we performed rescue experiments by overexpressing AK2 in IER5L-silenced MDA-MB-231 and BT-549 cells. Western blotting confirmed efficient IER5L knockdown and robust AK2 overexpression (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE). Functionally, IER5L silencing significantly reduced proliferation (CCK-8) and migration (Transwell), and ectopic AK2 expression partially restored both phenotypes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eF, G). These results indicate that AK2 contributes to IER5L-associated malignant behaviors, although the partial nature of the rescue suggests additional downstream pathways may also be involved.\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eIn this study, we systematically investigated the biological role of IER5L in breast cancer and identified an IER5L\\u0026ndash;AK2\\u0026ndash;associated regulatory axis that links metabolic regulation with malignant phenotypes. By integrating bioinformatic analyses, clinical tissue validation, in vitro and in vivo functional assays, and phosphoproteomic profiling, we provide multi-level evidence supporting the involvement of IER5L in breast cancer progression.\\u003c/p\\u003e \\u003cp\\u003eWe first evaluated the expression pattern and clinical relevance of IER5L. Our analyses demonstrated that IER5L is significantly upregulated in breast cancer tissues compared with normal breast tissues, and that higher IER5L expression is associated with poorer overall survival. Multivariate Cox regression analysis further indicated that IER5L expression serves as an independent prognostic factor. In addition, elevated IER5L levels were associated with younger patient age (\\u0026lt;\\u0026thinsp;60 years), more advanced clinical stage (II\\u0026ndash;IV), and higher histological grade (grade 3). These associations suggest that IER5L expression may increase with tumor aggressiveness and may be involved in both tumor development and progression.\\u003c/p\\u003e \\u003cp\\u003eIER5L is a member of the Immediate Early Response Genes (IERGs) family, which are rapidly induced in response to various cellular stress conditions, including DNA damage, chemotherapy exposure, and metabolic stress[\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]. Consistent with this functional background, our experimental data showed that IER5L knockdown significantly inhibited breast cancer cell proliferation and migration, promoted apoptosis, and suppressed tumor growth in xenograft models. These findings indicate that IER5L contributes to tumor cell survival and growth, potentially conferring an adaptive advantage under stress conditions commonly encountered in the tumor microenvironment.\\u003c/p\\u003e \\u003cp\\u003eAt the mechanistic level, we identified AK2 as an important downstream effector associated with IER5L-mediated regulation. AK2 is a mitochondrial intermembrane enzyme that plays a central role in cellular energy homeostasis by catalyzing the interconversion of ATP and AMP. Previous studies have shown that elevated lactylation of AK2 is associated with unfavorable prognosis in hepatocellular carcinoma[\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. Silencing IER5L led to a marked reduction in AK2 expression, and ectopic AK2 overexpression partially restored the proliferative capacity of IER5L-silenced cells. These results support a functional link between IER5L and AK2, although they do not establish direct regulation. Instead, the data suggest that AK2 acts as a downstream component within a broader IER5L-associated regulatory network.\\u003c/p\\u003e \\u003cp\\u003eBeyond AK2, protein kinase array analysis and subsequent Western blot validation revealed that IER5L influences the phosphorylation status of multiple signaling molecules, including STAT3, PRAS40, and YES, without substantially altering their total protein levels. These signaling components are closely connected to the JAK/STAT and mTOR pathways, which are known to integrate oncogenic signaling with metabolic control[\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. Together, these observations suggest that IER5L may function as an upstream regulatory node that coordinates metabolic regulation and proliferative signaling through multiple interconnected pathways.\\u003c/p\\u003e \\u003cp\\u003eBased on these findings, we propose a working model (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e) in which elevated IER5L expression is associated with enhanced mitochondrial energy metabolism via AK2 and increased activation of STAT3/mTOR-related signaling. Through this coordinated regulation, IER5L may contribute to metabolic reprogramming and support proliferative and migratory phenotypes in breast cancer cells. Importantly, this model represents a proposed regulatory framework derived from the experimental evidence presented and does not imply direct molecular interactions.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eSeveral limitations of the present study should be acknowledged. First, although our data demonstrate a functional association between IER5L and AK2, the precise molecular mechanisms underlying this relationship remain unclear and may involve intermediate regulators. Second, the potential impact of IER5L on the tumor immune microenvironment and on therapeutic resistance was not addressed and warrants further investigation. Third, while our analyses focused primarily on triple-negative breast cancer, whether the IER5L\\u0026ndash;AK2 axis operates similarly across other breast cancer subtypes requires validation in larger and more diverse patient cohorts.\\u003c/p\\u003e \\u003cp\\u003eFuture studies should therefore aim to (1) dissect the molecular mechanisms by which the IER5L\\u0026ndash;AK2 axis influences cellular energy metabolism, and (2) explore how IER5L-driven metabolic alterations affect immune responses and sensitivity to anticancer therapies. From a clinical perspective, our findings suggest that IER5L may serve as a prognostic biomarker and highlight the IER5L\\u0026ndash;AK2 axis as a potential target for combination therapeutic strategies in breast cancer.\\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eOur study indicates that IER5L is upregulated in breast cancer, especially TNBC, and is associated with poor prognosis. Functional and mechanistic analyses suggest that IER5L contributes to breast cancer progression through an AK2-associated signaling axis linked to STAT3/mTOR activation. These findings support IER5L as a potential prognostic biomarker and therapeutic target.\\u003c/p\\u003e\"},{\"header\":\"Methods and Materials\",\"content\":\"\\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eData sources and bioinformatic analyses\\u003c/h2\\u003e \\u003cp\\u003eGene expression profiles of breast cancer and normal breast tissues were obtained from the GEO database (GSE54002) and analyzed using R. Data were normalized, and differential expression was assessed using standard pipelines. Prognostic analyses were performed using the METABRIC cohort (downloaded via cBioPortal), including clinical variables such as age, stage, and histological grade. Expression and survival associations were further validated using the GEPIA3.0 and TIMER3.0 online platforms.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCell culture and lentiviral transduction\\u003c/h2\\u003e \\u003cp\\u003eMCF-10A human mammary epithelial cells and breast cancer cell lines (MDA-MB-231, MCF-7, BT-549) were purchased from the cell bank of Shanghai Mingjin Biotechnology Co., Ltd., and cultured according to the supplier\\u0026rsquo;s instructions. Lentiviral shRNAs targeting IER5L were used to generate stable knockdown cell lines. Cells transduced with non-targeting shRNA served as controls. For rescue experiments, AK2 was ectopically overexpressed in IER5L-silenced cells using lentiviral constructs.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eQuantitative real-time PCR (qRT-PCR)\\u003c/h2\\u003e \\u003cp\\u003eTotal RNA was isolated using TRIzol. RNA concentration and purity were assessed using a NanoDrop spectrophotometer. cDNA was synthesized from 1 \\u0026micro;g of total RNA using Hiscript QRT Supermix for qPCR (gDNA WIPER). qRT-PCR was performed using SYBR Green mastermix (Vazyme Q111-02) on an ABI 7900 system. Melting curve analysis was used to confirm amplification specificity. GAPDH served as an internal reference.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eWestern blotting\\u003c/h2\\u003e \\u003cp\\u003eCells were lysed in RIPA buffer containing PMSF. Protein samples were denatured in loading buffer, separated by SDS\\u0026ndash;PAGE, and transferred to PVDF membranes. Membranes were blocked in 5% non-fat milk in TBST and incubated with primary antibodies at 4\\u0026deg;C overnight (IER5L 1:1000; β-actin 1:4000). After washing, membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Signals were visualized using ECL substrate and quantified with β-actin or GAPDH as loading controls.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eImmunohistochemistry (IHC) and H\\u0026amp;E staining\\u003c/h2\\u003e \\u003cp\\u003eParaffin sections were deparaffinized, rehydrated, and subjected to antigen retrieval using citrate or EDTA buffer. Endogenous peroxidase was blocked with 3% H₂O₂, and non-specific binding was blocked with goat serum. Sections were incubated with primary antibodies, followed by secondary antibody incubation and DAB development. Slides were counterstained with hematoxylin, dehydrated, and mounted. H\\u0026amp;E staining followed standard procedures.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCell proliferation, colony formation, apoptosis, and migration assays\\u003c/h2\\u003e \\u003cp\\u003eFor CCK-8 assays, equal numbers of cells were seeded in 96-well plates and assessed daily by measuring absorbance at 450 nm. Colony formation assays were performed by seeding 500\\u0026ndash;1000 cells per well in 6-well plates and culturing for 10\\u0026ndash;14 days, followed by fixation and crystal violet staining. Apoptosis was evaluated using Annexin V-FITC/PI staining and flow cytometry. Migration was assessed using Transwell chambers without Matrigel; migrated cells were fixed, stained, and quantified after 24 h.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eXenograft mouse model and in vivo imaging\\u003c/h2\\u003e \\u003cp\\u003eBALB/c nude mice (4\\u0026ndash;6 weeks old) were purchased from Shanghai Yaokang Biotechnology Co., Ltd. and used for xenograft experiments. Mice were allocated to experimental groups according to the study design, with an average body weight of 17.75 g in the experimental group and 17.85 g in the control group at the beginning of the experiments. Mice were subcutaneously injected with MDA-MB-231 cells expressing control shRNA or shIER5L. Tumor growth was monitored at predefined time points, and in vivo fluorescence imaging was performed to assess tumor burden. Tumors were excised, weighed, and processed for western blotting and immunohistochemistry (IHC). All procedures were performed under inhalational anesthesia using isoflurane, and humane endpoints were applied to minimize animal suffering. At the end of the experiments, mice were euthanized by CO₂ inhalation followed by cervical dislocation in accordance with institutional guidelines.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eTissue microarray cohort and ethics\\u003c/h2\\u003e \\u003cp\\u003eTNBC tissue microarrays (150 TNBC and 30 adjacent tissues) were purchased from Shanghai Mijian Biotechnology Co., Ltd. Ethical approval was provided by the supplier\\u0026rsquo;s ethics committee (Ethics Approval No.: SHYJS-CP-2210009).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eProteomic analysis and candidate selection\\u003c/h2\\u003e \\u003cp\\u003eProteins downregulated after IER5L knockdown were identified by iTRAQ-based proteomic profiling and cross-referenced with TCGA analyses for expression and prognostic relevance in TNBC. Candidate proteins were selected using the following criteria: differential protein logFC\\u0026thinsp;\\u0026lt;\\u0026thinsp;\\u0026minus;\\u0026thinsp;0.6; breast cancer expression log2FoldChange.exp\\u0026thinsp;\\u0026gt;\\u0026thinsp;1; HR.val.optimal.os\\u0026thinsp;\\u0026gt;\\u0026thinsp;1; and P.val.optimal.os\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05. CNN3, AK2, and HMGCS1 were prioritized for validation.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical analysis\\u003c/h2\\u003e \\u003cp\\u003eData analyses were performed using RStudio and SPSS 27. Graphs were generated using Prism 9. Unless otherwise stated, data are presented as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SD from at least three independent experiments. Two-group comparisons were performed using appropriate parametric or non-parametric tests. A two-sided P value\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 was considered statistically significant.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003ch2\\u003eAnimal reporting and ethical approval\\u003c/h2\\u003e\\n\\u003cp\\u003eAll animal experiments were conducted in accordance with institutional guidelines and approved by the Animal Ethics Committee of Sichuan Provincial People\\u0026rsquo;s Hospital (Approval No.AF/SW-05/01.1). This study is reported in compliance with the ARRIVE guidelines. BALB/c nude mice (4\\u0026ndash;6 weeks old) were used for xenograft experiments and were allocated to experimental groups according to the study design. Tumor growth was monitored at predefined time points, and humane endpoints were applied to minimize suffering. At the end of the experiments, mice were euthanized by CO₂ inhalation followed by cervical dislocation in accordance with institutional guidelines.\\u003c/p\\u003e\\n\\u003ch2\\u003eEthics approval and consent to participate\\u003c/h2\\u003e\\n\\u003cp\\u003eHuman tissue microarray samples (150 triple-negative breast cancer tissues and 30 adjacent non-tumor tissues) were purchased from Shanghai Xinchao Biotechnology Co.Ltd. Ethical approval for the use of these samples was obtained from the supplier\\u0026rsquo;s ethics committee (Ethics Approval No.SHYJS-CP-2210009). All samples were provided in a de-identified form. The study was conducted in accordance with the Declaration of Helsinki and relevant institutional guidelines and regulations. Informed consent was obtained by the supplier from the participants or was waived by the ethics committee in accordance with applicable regulations.\\u003c/p\\u003e\\n\\u003ch2\\u003eCompeting Interests\\u003c/h2\\u003e\\n\\u003cp\\u003eThe authors declare that they have no competing interests. The corresponding authors confirm that they are responsible for submitting this declaration on behalf of all authors.\\u003c/p\\u003e\\n\\u003ch2\\u003eFunding\\u003c/h2\\u003e\\n\\u003cp\\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\\u003c/p\\u003e\\n\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\n\\u003cp\\u003eY.L. and X.W. conceived and supervised the study.X.Z. performed the experiments and drafted the manuscript.J.Z. assisted with the experimental work.All authors reviewed and approved the final manuscript.\\u003c/p\\u003e\\n\\u003ch2\\u003eAcknowledgements\\u003c/h2\\u003e\\n\\u003cp\\u003eThe authors thank all patients who contributed tissue samples to this study. We acknowledge the technical support provided by the experimental platform staff at the School of Medicine, University of Electronic Science and Technology of China, and the assistance of the animal facility staff during the in vivo experiments. We also thank the contributors of the GEO, METABRIC, TCGA, GEPIA, and TIMER databases for making their datasets publicly available.\\u003c/p\\u003e\\n\\u003ch2\\u003eData Availability\\u003c/h2\\u003e\\n\\u003cp\\u003ePublicly available datasets were used in this study, including GEO (GSE54002) and METABRIC (accessed via cBioPortal). Analyses were also performed using the GEPIA3.0 and TIMER3.0 online platforms. The datasets generated and/or analyzed during the current study, including raw and processed data supporting the findings, are available from the corresponding author upon reasonable request.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eF. 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Sakurai, \\u0026ldquo;PP2A‐B55 and its adapter proteins IER2 and IER5 regulate the activity of RB family proteins and the expression of cell cycle‐related genes,\\u0026rdquo; \\u003cem\\u003eThe FEBS Journal\\u003c/em\\u003e, vol. 290, no. 3, pp. 745\\u0026ndash;762, Feb. 2023, doi: 10.1111/febs.16612.\\u003c/li\\u003e\\n\\u003cli\\u003eL. Kyjacova \\u003cem\\u003eet al.\\u003c/em\\u003e, \\u0026ldquo;IER2-induced senescence drives melanoma invasion through osteopontin,\\u0026rdquo; \\u003cem\\u003eOncogene\\u003c/em\\u003e, vol. 40, no. 47, pp. 6494\\u0026ndash;6512, Nov. 2021, doi: 10.1038/s41388-021-02027-6.\\u003c/li\\u003e\\n\\u003cli\\u003eJ. R. Crespo \\u003cem\\u003eet al.\\u003c/em\\u003e, \\u0026ldquo;The PP2A regulator IER5L supports prostate cancer progression,\\u0026rdquo; \\u003cem\\u003eCell Death Dis\\u003c/em\\u003e, vol. 15, no. 7, p. 514, Jul. 2024, doi: 10.1038/s41419-024-06907-z.\\u003c/li\\u003e\\n\\u003cli\\u003eR. Cao \\u003cem\\u003eet al.\\u003c/em\\u003e, \\u0026ldquo;Molecular mechanism of PP2A/B55\\u0026alpha; phosphatase inhibition by IER5,\\u0026rdquo; \\u003cem\\u003eCell Chemical Biology\\u003c/em\\u003e, vol. 32, no. 4, pp. 631-642.e7, Apr. 2025, doi: 10.1016/j.chembiol.2025.03.004.\\u003c/li\\u003e\\n\\u003cli\\u003eJ. Krishnaraj \\u003cem\\u003eet al.\\u003c/em\\u003e, \\u0026ldquo;IER5 Promotes Ovarian Cancer Cell Proliferation and Peritoneal Dissemination,\\u0026rdquo; \\u003cem\\u003eCancers\\u003c/em\\u003e, vol. 17, no. 4, p. 610, Feb. 2025, doi: 10.3390/cancers17040610.\\u003c/li\\u003e\\n\\u003cli\\u003eS. Kawabata, Y. Ishita, Y. Ishikawa, and H. Sakurai, \\u0026ldquo;Immediate‐early response 5 (IER5) interacts with protein phosphatase 2A and regulates the phosphorylation of ribosomal protein S6 kinase and heat shock factor 1,\\u0026rdquo; \\u003cem\\u003eFEBS Letters\\u003c/em\\u003e, vol. 589, no. 23, pp. 3679\\u0026ndash;3685, Nov. 2015, doi: 10.1016/j.febslet.2015.10.013.\\u003c/li\\u003e\\n\\u003cli\\u003eW. Wu \\u003cem\\u003eet al.\\u003c/em\\u003e, \\u0026ldquo;Identification of immediate early response protein 2 as a regulator of angiogenesis through the modulation of endothelial cell motility and adhesion,\\u0026rdquo; \\u003cem\\u003eInternational Journal of Molecular Medicine\\u003c/em\\u003e, vol. 36, no. 4, pp. 1104\\u0026ndash;1110, Oct. 2015, doi: 10.3892/ijmm.2015.2310.\\u003c/li\\u003e\\n\\u003cli\\u003eT. Ueda, Y. Kohama, and H. Sakurai, \\u0026ldquo;IER family proteins are regulators of protein phosphatase PP2A and modulate the phosphorylation status of CDC25A,\\u0026rdquo; \\u003cem\\u003eCellular Signalling\\u003c/em\\u003e, vol. 55, pp. 81\\u0026ndash;89, Mar. 2019, doi: 10.1016/j.cellsig.2018.12.012.\\u003c/li\\u003e\\n\\u003cli\\u003eZ. Yang \\u003cem\\u003eet al.\\u003c/em\\u003e, \\u0026ldquo;Lactylome analysis suggests lactylation-dependent mechanisms of metabolic adaptation in hepatocellular carcinoma,\\u0026rdquo; \\u003cem\\u003eNat Metab\\u003c/em\\u003e, vol. 5, no. 1, pp. 61\\u0026ndash;79, Jan. 2023, doi: 10.1038/s42255-022-00710-w.\\u003c/li\\u003e\\n\\u003cli\\u003eF. Cai \\u003cem\\u003eet al.\\u003c/em\\u003e, \\u0026ldquo;AK2 Promotes the Migration and Invasion of Lung Adenocarcinoma by Activating TGF-\\u0026beta;/Smad Pathway In vitro and In vivo,\\u0026rdquo; \\u003cem\\u003eFront. Pharmacol.\\u003c/em\\u003e, vol. 12, p. 714365, Sep. 2021, doi: 10.3389/fphar.2021.714365.\\u003c/li\\u003e\\n\\u003cli\\u003e\\u0026ldquo;cancer.\\u0026rdquo;Accessed:Sep.14,2025.[Online].Available: https://www.proteinatlas.org/ENSG00000004455-AK2/cancer\\u003c/li\\u003e\\n\\u003cli\\u003eL. Alberghina, \\u0026ldquo;The Warburg Effect Explained: Integration of Enhanced Glycolysis with Heterogeneous Mitochondria to Promote Cancer Cell Proliferation,\\u0026rdquo; \\u003cem\\u003eIJMS\\u003c/em\\u003e, vol. 24, no. 21, p. 15787, Oct. 2023, doi: 10.3390/ijms242115787.\\u003c/li\\u003e\\n\\u003cli\\u003eI. Barba, L. Carrillo-Bosch, and J. Seoane, \\u0026ldquo;Targeting the Warburg Effect in Cancer: Where Do We Stand?,\\u0026rdquo; \\u003cem\\u003eIJMS\\u003c/em\\u003e, vol. 25, no. 6, p. 3142, Mar. 2024, doi: 10.3390/ijms25063142.\\u003c/li\\u003e\\n\\u003cli\\u003eM. Liao \\u003cem\\u003eet al.\\u003c/em\\u003e, \\u0026ldquo;Targeting the Warburg effect: A revisited perspective from molecular mechanisms to traditional and innovative therapeutic strategies in cancer,\\u0026rdquo; \\u003cem\\u003eActa Pharmaceutica Sinica B\\u003c/em\\u003e, vol. 14, no. 3, pp. 953\\u0026ndash;1008, Mar. 2024, doi: 10.1016/j.apsb.2023.12.003.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Triple-negative breast cancer, IER5L, AK2, Metabolic reprogramming, STAT3/mTOR signaling\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8661639/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8661639/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eBreast cancer is the most frequently diagnosed malignancy in women worldwide. Triple-negative breast cancer (TNBC) has particularly poor outcomes, largely due to the lack of effective therapeutic targets. Here, we investigated the expression pattern, functional relevance, and potential molecular mechanisms of Immediate Early Response 5-Like gene (IER5L) in breast cancer by integrating public database analyses, clinical tissue validation, in vitro assays, and in vivo xenograft experiments. IER5L was significantly upregulated in breast cancer tissues, with the highest levels observed in TNBC, and elevated IER5L expression was associated with unfavorable prognosis. Silencing IER5L suppressed proliferation and migration of breast cancer cells, increased apoptosis, and inhibited tumor growth in nude mouse xenograft models. Mechanistically, proteomic profiling identified adenylate kinase 2 (AK2) as a key downstream effector of IER5L. IER5L depletion led to reduced AK2 expression and attenuation of STAT3/mTOR-related signaling. Importantly, rescue experiments demonstrated that ectopic AK2 expression partially reversed the inhibitory effects of IER5L knockdown on cell proliferation and migration. Together, these findings suggest that IER5L contributes to breast cancer progression through an AK2-associated STAT3/mTOR signaling program and support IER5L as a potential prognostic biomarker and therapeutic target, particularly in TNBC.\\u003c/p\\u003e\",\"manuscriptTitle\":\"The IER5L–AK2 axis drives aggressive behavior in triple-negative breast cancer\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-02-11 15:49:55\",\"doi\":\"10.21203/rs.3.rs-8661639/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"reviewerAgreed\",\"content\":\"114868132875865254305439349420399394240\",\"date\":\"2026-05-16T12:24:38+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"180757478346933643072513834741899119723\",\"date\":\"2026-05-15T02:53:57+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2026-02-06T12:54:09+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2026-02-06T12:49:34+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2026-02-05T09:15:14+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2026-01-31T06:45:57+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Scientific Reports\",\"date\":\"2026-01-31T06:36:31+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"2a4405ab-3708-4b4c-97ef-10b8d9a520a0\",\"owner\":[],\"postedDate\":\"February 11th, 2026\",\"published\":true,\"recentEditorialEvents\":[{\"type\":\"reviewerAgreed\",\"content\":\"114868132875865254305439349420399394240\",\"date\":\"2026-05-16T12:24:38+00:00\",\"index\":180,\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"180757478346933643072513834741899119723\",\"date\":\"2026-05-15T02:53:57+00:00\",\"index\":179,\"fulltext\":\"\"}],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[{\"id\":62751406,\"name\":\"Health sciences/Biomarkers\"},{\"id\":62751407,\"name\":\"Biological sciences/Cancer\"},{\"id\":62751408,\"name\":\"Health sciences/Oncology\"}],\"tags\":[],\"updatedAt\":\"2026-02-11T15:49:55+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-02-11 15:49:55\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8661639\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8661639\",\"identity\":\"rs-8661639\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}