ASPSCR1 promotes osteosarcoma progression through the upregulation of glycolysis

ASPSCR1 promotes osteosarcoma progression through the upregulation of glycolysis | 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 ASPSCR1 promotes osteosarcoma progression through the upregulation of glycolysis Hanbing Song, Fei Wang, Linqin He, Shuying Wu, Youyou Li, Wenqi Zhang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8446485/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Osteosarcoma (OS), the most common primary bone malignancy, is highly metastatic and difficult to treat. Although ASPSCR1 is implicated in multiple cancers, its role and mechanism in OS remain unclear. This study aims to clarify ASPSCR1 function in OS progression.ASPSCR1 expression and prognosis were evaluated via immunohistochemistry and statistics in clinical cohorts. Lentiviral shRNA-mediated ASPSCR1 knockdown in OS cells enabled functional assessments of proliferation, apoptosis, migration/invasion. Gene Set Enrichment Analysis (GSEA) identified pathways enrichment, further corroborated by glucose, lactate, and ATP assays. In vivo tumorigenicity was assessed using subcutaneous xenograft.ASPSCR1 was overexpressed in OS tissues and associated with poor survival. ASPSCR1 knockdown inhibited proliferation and migration while inducing apoptosis in OS cells. GSEA revealed significant enrichment of ASPSCR1-associated genes in the glycan biosynthesis. PKM2 inhibition (PKM2-IN-3) abolished ASPSCR1-driven glycolytic activity and proliferation. Consistent with in vitro findings, in vivo experiments robustly supported the pivotal role of ASPSCR1 knockdown in reducing glycolytic activity and inhibiting OS tumor growth. ASPSCR1 promotes OS progression via upregulating glycolysis, representing a potential therapeutic target. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Osteosarcoma (OS), a highly malignant connective tissue tumor, ranks significantly in primary bone malignancies, posing severe threats to patients' lives and well-being. Characterized by tumor cells directly producing bone and osteoid tissues, it exhibits high malignancy, poor prognosis, and rapid progression with a tendency to metastasize to the lungs and other distant sites 1 . Current treatments primarily involve surgical resection combined with chemotherapy and radiotherapy 2 . In recent years, targeted therapies have shown remarkable progress, offering new hope for improving patient outcomes 3 . However, challenges such as limited drug options, high costs, and drug resistance persist 4 . Therefore, exploring the mechanisms underlying OS development is crucial for developing novel and effective targeted therapies. Glycolysis is a crucial metabolic pathway for tumor cells to acquire energy, particularly in hypoxic or rapidly proliferating tumor environments 5 . This metabolic reprogramming not only provides essential energy and biosynthetic precursors for tumor cells but also alters the tumor microenvironment, promoting invasion and metastasis 6 . In OS, the rapid growth and uncontrolled characteristics of tumor cells make them highly dependent on glucose uptake and glycolysis 7 . Therefore, developing targeted therapeutic strategies for OS from the perspective of glycolysis is of great theoretical and practical significance. ASPSCR1, fullname is 'ASPSCR1 tether for SLC2A4, UBX domain containing', also kown as TUG, which sequesters the GLUT4 in intracellular vesicles in muscle and fat cells in the absence of insulin, and redistributes the GLUT4 to the plasma membrane within minutes of insulin stimulation 8, 9 . It shuttles between the nucleus and cytoplasm, regulating gene transcription and protein synthesis 10 . Notably, the ASPSCR1::TFE3 fusion protein, formed by the fusion of ASPSCR1 with TFE3, leverages TFE3's basic helix-loop-helix (bHLH) domain to drive oncogenic transcription. This fusion protein regulates genes involved in lysosomal function, autophagy, and angiogenesis, serving as a central genetic driver in alveolar soft part sarcoma (ASPS) and Xp11 translocation renal cell carcinoma (RCC) 11 . Research indicates that ASPSCR1::TFE3 preferentially binds to super-enhancers, promoting angiogenesis and supporting tumor growth. In TFE3-rearranged RCC, patients with ASPSCR1-TFE3 fusion exhibit better responses to immunotherapy combinations 12 . ASPSCR1's somatic variations are also linked to poor prognosis in hereditary diffuse gastric cancer (HDGC) 13 . Moreover, ASPSCR1 mitigates oxidative stress, proliferation, and migration of cholangiocarcinoma cells induced by rCsNOSIP from Opisthorchis felineus 14 . These findings highlight ASPSCR1's complex roles across different cancer contexts. In this study, we aim to comprehensively explore ASPSCR1's role and mechanisms in OS. We will analyze and validate ASPSCR1 expression in OS tissues and cell lines, knock down ASPSCR1 expression, and investigate its effects on cell proliferation, migration, invasion, and glycolysis. Our goal is to uncover ASPSCR1's functions and mechanisms in OS, particularly focusing on its downstream mechanism in glycolysis regulation. This research will provide new insights into OS pathogenesis and may identify novel therapeutic targets for treatment. 2. Materials and methods 2.1 Tissue Samples OS tissue sections (n=80, model: LBN802) were provided by Xi'an Tebos Pharmaceutical Technology Co., Ltd. Additionally, normal bone tissue sections (n=9, model: YBRHost09-M002) were obtained from Shanghai YiBeiRui Bioscience Co., Ltd. The tumor samples were collected from patients with primary OS who underwent surgical resection. All samples were evaluated by pathologists andparaffin-embedded. Patient data included sex, age, and other clinical parameters. Written informed consent was obtained from all subjects, and human tissue collection was approved by the Ethics Committee of the First Affiliated Hospital, Heilongjiang University of Chinese Medicine (Approval number: HZYLLKT202341601). 2.2 Immunohistochemistry (IHC) and Hematoxylin-Eosin (HE) staining Paraffin-embedded tissue samples sections were subjected to IHC analysis for ASPSCR1, Ki67, and GLUT1 proteins. Tissue sections were heated at 60°C for 2 hours, deparaffinized with xylene, and rehydrated through a descending ethanol series. Sections were then incubated with primary antibodies against ASPSCR1 (PhD Bio, 1:100, M05168), Ki67 (Abcam, 1:200, ab16667), and GLUT1 (PhD Bio, 1:100, BA3481-2) at 4°C for 12 hours. The following day, sections were washed with PBS and incubated with a goat anti-rabbit IgG H&L (HRP) pre-adsorbed secondary antibody (Abcam, 1:200, ab97080) at room temperature for 2 hours. Staining was visualized using the ImmunoPure Metal-Enhanced DIY ELISA Builder (DAB) substrate kit according to the manufacturer's instructions, and results were observed under a light microscope. Scoring of staining intensity and the percentage of positive cells was performed by two pathologists. The IHC score was calculated as the sum of the intensity and percentage scores. Tumor tissue sections were also subjected to HE staining. Sections were fixed in 95% ethanol, washed, stained with hematoxylin for nuclear staining, followed by eosin for cytoplasmic staining, and then air-dried. 2.3 Cell lines and Cultures Human OS cell lines (U2OS, SW-1353, HOS, 143B) and normal human osteoblasts (Chondrocytes) were obtained from the Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). OS cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM, Biological Industries, Israel) supplemented with 10% fetal bovine serum (FBS, Ausbian VS500T), 1% streptomycin, and 1% penicillin. Chondrocyte cells were cultured in DMEM/F12. All cells were maintained at 37°C with 5% CO₂ in a humidified incubator.All cell lines used in this study were authenticated via STR profiling and verified as mycoplasma-free. 2.4 Lentivirus Production and Infection (RNAi) Lentiviral vectors containing shRNA sequences targeting ASPSCR1 and negative control shRNA were obtained from Shanghai YiBeiRui BioPharmaceuticals Co., Ltd. U2OS and HOS cells were seeded in 6-well plates at 2×10⁵ cells per well. After 24 hours, cells were infected with lentivirus following the manufacturer's instructions. Following a 72-hour incubation, cells were treated with fresh medium containing 10 µg/mL puromycin for selection. The sequences of used in this study are listed as follows: shASPSCR1-1: 5'-CCTGCACCTAAGTCTGAGCCA-3'; shASPSCR1-2: 5'-GCCTGATGAGTTCTTTGAGCT-3'; shASPSCR1-3: 5'-TCGAGGTTGCAGGACTCTTTC-3'. 2.5 Quantitative real-time Polymerase Chain Reaction (qPCR) Total RNA was isolated from OS tissues and cells using TRIzol reagent (Sigma, T9424-100mL) according to the manufacturer's instructions. cDNA was synthesized using the HiScript QRT SuperMix Kit (Vazyme, R123-01) and stored at -80°C. The following primer pairs were used: GAPDH forward, 5'-TGACTTCAACAGCGACACCCA-3'; GAPDH reverse, 5'-CACCCTGTTGCTGTAGCCAAA-3'; ASPSCR1 forward, 5'-AACATGGTTCGCATCGCTTTG-3'; ASPSCR1 reverse, 5'-ACCCGTCACCTCATCCCTC-3'. 2.6 Western Blot (WB) Cells were washed once with cold phosphate-buffered saline (PBS) and lysed in RIPA lysis buffer for 10 minutes. They were then centrifuged at 12,000 rpm for 10 minutes at 4°C to obtain the supernatant. Equal amounts of protein were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Millipore, USA). The membranes were blocked using 5% skimmed milk for 2 hours and then incubated with primary antibodies overnight at 4°C. The primary antibodies used were anti-ASPSCR1 (1:1000, BOSTER), GAPDH (1:30,000, Proteintech), PKM2 (1:5000, Proteintech), HK2 (1:3000, Proteintech), GLUT1 (1:1000, Proteintech), ADH4 (1:2000, Abcam), ALDOC (1:5000, Proteintech), ALDOA (1:3000, Proteintech), G6PD (1:1000, Abcam), FBP1 (1:2000, Proteintech), ADH6 (1:5000, Proteintech), LDHA (1:3000, Abcam), and β-Actin (1:4000, Proteintech). After incubation with appropriate anti-mouse/rabbit secondary antibodies for 1 hour at room temperature, immunoreactive bands were detected using a chemiluminescence kit (Millipore, USA). 2.7 Cell Proliferation Assay Cell proliferation was assessed using the Cell Counting Kit-8 (Sigma, 96992). U2OS and HOS cells (1 × 10³ cells/well) were seeded in 96-well plates (Corning, 3596) and cultured for 24, 48, and 72 hours. Each well received 10 μL of CCK-8 solution, followed by incubation at 37°C with 5% CO₂ for 2 hours. Absorbance at 450 nm was measured using a microplate reader (Tecan Infinite, M2009PR). 2.8 Colony Formation Assay U2OS and HOS cells with ASPSCR1 knockdown or overexpression were seeded in 6-well plates at 500 cells per well 5 days post-infection. Cells were cultured for 8 days with medium changes every 3 days. Cell colonies were photographed under a fluorescence microscope (Olympus, IX71), fixed with 4% paraformaldehyde (Sinopharm Chemical Reagent Co., Ltd., AR Shanghai Test) for 30 minutes, and stained with Giemsa solution (Shanghai Dingguo Biotechnology Co., Ltd., AR-0752) for 30 minutes. Colonies were counted after air-drying and washing with D-Hanks. 2.9 Flow Cytometry Apoptosis was assessed using the Annexin V-FITC/Propidium Iodide (PI) Apoptosis Detection Kit (eBioscience, 88-8007-7). Transfected cells were trypsinized, collected, and washed twice with cold 1× PBS. Cells were suspended in 400 μL 1× binding buffer, stained with 5 μL Annexin V-FITC for 15 minutes at 4°C in the dark, followed by 10 μL PI for 10 minutes. Stained cells were analyzed by flow cytometry to determine early and late apoptosis. 2.10 Scratch Assay U2OS and HOS cells were seeded in 6-well plates with serum-enriched medium (Corning, 10-013-CVR). Upon reaching over 90% confluence, a scratch was made using a scratch tool (VP Scientific, VP408FH). After washing with D-Hanks, cells were cultured in fresh DMEM (Corning, 10-013-CVR) with 2% FBS. Scratch area changes were observed under a microscope (Olympus, IX73) at 0 h and 24 h. Cellomics (Thermo, ArrayScan VT1) quantified the migrated area. 2.11 Transwell Assay Cell invasion was assessed using Transwell plates (Corning, 3422). Logarithmic growth phase cells were resuspended in serum-free medium and seeded in the upper chamber coated with Matrigel. The lower chamber contained 600 μL of complete medium with 30% fetal bovine serum. After 24 hours of incubation, cells were stained and counted. 2.12 Measurement of Glycolysis Levels (GLU, LA, ATP) The levels of glucose (GLU), lactic acid (LA), and adenosine triphosphate (ATP) were measured to evaluate glycolysis activity. All assays were performed in triplicate and the results were normalized to protein concentration or cell number to ensure accuracy and reproducibility. For glucose consumption assay, GLU concentration was determined using the o-toluidine method with a glucose assay kit (Solarbio, Beijing, China, product number: BC2500, specification: 50T/48S). Cells were cultured in 96-well plates at a density of 5×10³ cells per well and incubated at 37°C for 48 hours. The absorbance was measured at 505 nm to calculate glucose consumption levels. For ATP content assay, ATP levels were measured using an ATP content assay kit (Solarbio, Beijing, China, product number: BC0300, specification: 50T/48S). Cells were seeded in 96-well plates at 5×10³ cells per well. Cell extracts were prepared and analyzed according to the manufacturer's instructions. The absorbance at 340 nm was measured to determine ATP content. For LA production assay, LA production was measured using a LA content assay kit (Solarbio, Beijing, China, product number: BC2230, specification: 50T/24S). Cells were seeded in 96-well plates at 5×10³ cells per well. Cell extracts were prepared and analyzed according to the manufacturer's instructions. The absorbance at 570 nm was measured to determine LA content. 2.13 Tumorigenicity Assay Twelve female BALB/c nude mice (4-6 weeks old) were purchased from Jiangsu Jicui Pharmachem Bio-technology Co. Ltd. The mice were housed under pathogen-free conditions. HOS cells (1×10⁷ cells in 200 μL) were injected subcutaneously into the right flank of each mouse. The mice were randomly divided into two groups: negative control (NC) and knockdown (KD). Tumor growth was measured at specified time points (7, 11, 14, 18, and 21 days) using calipers, and tumor volume was calculated using the formula (π/6) × L× W², where L is the tumor length and W is the tumor width. Mice were euthanized 25 days post-injection, and tumor weights were measured. Tissues were stored at -80°C for subsequent analysis. All animal experiments were approved by the Ethics Committee of The First Affiliated Hospital of Heilongjiang University of Traditional Chinese Medicine (Approval number: HZYLLKT202341601) and conducted in accordance with their guidelines and rules. 2.14 Statistical Methods All data are presented as the mean ± standard error of the mean (SEM) and were analyzed using GraphPad Prism 8.0 (La Jolla, USA). Differences between groups were assessed using Student's t-test or one-way analysis of variance (ANOVA). All statistical tests were two-sided, and a p-value < 0.05 was considered statistically significant. 3. Results 3.1 ASPSCR1 is highly expressed in OS and is associated with poor prognosis To gain a comprehensive understanding of ASPSCR1's role in OS, we analyzed data from the GEO database (GSE42352) and Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database. Our analysis revealed that ASPSCR1 expression levels were significantly higher in OS tissues than in normal tissues (Figure 1A) . We then categorized the samples based on the media2n expression value of ASPSCR1, dividing them into high-expression and low-expression groups. Kaplan-Meier survival analysis with the log-rank test demonstrated that patients with high ASPSCR1 expression had significantly shorter overall survival compared to those with low expression (hazard ratio (HR) = 2.88 (95% CI: 1.29-6.44), P = 0.00707) (Figure 1B) . A Cox multivariate regression analysis was performed to evaluate the association between the expression of ASPSCR1 and clinical characteristics. These findings suggest that the expression level of ASPSCR1 can serve as an independent prognostic factor (HR=2.62 (95% CI: 1.59-4.34), P = 0.000174), irrespective of other clinical indicators (Figure 1C) . To gain a profound understanding of the clinical significance of ASPSCR1 in OS, we constructed tissue microarrays using normal and tumor tissues from OS patients and performed HE staining and IHC analysis. By comparing the staining patterns and conducting quantitative analysis (Table 1) , we found that ASPSCR1 expression was significantly elevated in OS tissues compared to normal tissues (Figure 1D) . To delve deeper into the relationship between ASPSCR1 expression levels and the clinicopathological characteristics of OS, we employed statistical methods for an in-depth analysis of the data. Initially, we classified samples into ASPSCR1 high-expression and low-expression groups, using the median expression value across all OS samples as the threshold. Subsequently, based on patients' clinical information, we utilized the Mann-Whitney U test to reveal a significant positive correlation between ASPSCR1 expression levels and both gender and age of the patients (Table 2) . To further validate this result, we conducted a Pearson rank correlation analysis, which again demonstrated that ASPSCR1 expression levels increased correspondingly with the advancement of tumor malignancy in patients (Table 3) . Our above statistical analyses further confirmed that ASPSCR1 expression levels are closely associated with tumor malignancy and patient outcomes, highlighting its importance in the clinical management of OS. 3.2 The construction of ASPSCR1 knockdown in OS cells To elucidate the specific role of ASPSCR1 in the phenotypic traits and biological functions of OS cells, we meticulously designed and executed a comprehensive series of cellular-level experiments. Initially, we screened various OS cell lines for their ASPSCR1 expression levels using qPCR technology. Based on this screening, two cell lines, HOS and U2OS, which exhibited relatively high ASPSCR1 expression, were selected for further experimentation (Figure 2A) . To establish effective HOS and U2OS cell models, we identified suitable interference targets through a rigorous process involving qPCR and WB analyses (Figure 2B, C) . Guided by these screening results, we meticulously selected the two shASPSCR1 sequences that demonstrated the highest knockdown efficiency, with the aim of developing an OS cell model characterized by downregulated ASPSCR1 expression. Subsequently, we underwent a series of meticulous construction and validation procedures. These included observing the infection efficiency under a green fluorescent protein microscope, which confirmed that the cell infection efficiency surpassed 80% and maintained good cell health (Figure 2D) . Furthermore, we conducted double validation of the knockdown efficiency through additional analyses (Figure 2E, F) . Following these rigorous steps, we successfully constructed HOS and U2OS cell models featuring dual-target knockdown of ASPSCR1. 3.3 Knockdown of ASPSCR1 inhibits the progression of OS cells In subsequent functional validation experiments, we employed the CCK8 Assay to observe that the knockdown of ASPSCR1 significantly impaired the proliferative capacity of HOS and U2OS cells (Figure 3A). Furthermore, ASPSCR1-knockdown cells exhibited a reduced clone formation ability, forming smaller and fewer clones compared to control cells (Figure 3B). These observations underscore the pivotal role of ASPSCR1 in facilitating cell growth. Additionally, flow cytometry analysis revealed that the reduction of ASPSCR1 expression augmented the apoptotic tendency of HOS and U2OS cells (Figure 3C), further emphasizing its crucial role in regulating cellular fate. To comprehensively assess the impact of ASPSCR1 on the migratory and invasive capabilities of OS cells, we conducted scratch and Transwell experiments. The results demonstrated that ASPSCR1 knockdown markedly inhibited the migration and invasion of OS cells (Figure 3D, E), providing compelling evidence for its potential role in the malignant progression of OS. In summary, our research findings not only unveiled the abnormally elevated expression of ASPSCR1 in OS cells but also validated its essential role in promoting cell proliferation, inhibiting apoptosis, and enhancing cellular migration and invasion abilities through a series of rigorous functional experiments. 3.4 Knockdown of ASPSCR1 inhibits glycolysis levels in OS cells In our investigation of the molecular mechanisms underlying OS, we discovered that the high expression of ASPSCR1 was significantly enriched in the glycan biosynthesis, suggesting its potential role in tumor glucose metabolism (Figure 4A) . To substantiate this observation, we conducted further experimental validations, which revealed that the knockdown of ASPSCR1 markedly impaired the glycolytic capacity of HOS and U2OS cells. This was evident from decreased glucose uptake, reduced ATP levels, and limited lactate production (Figure 4B) , all of which serve as direct indicators of diminished glycolysis activity. Concurrently, we observed that multiple crucial proteins involved in the glycolysis pathway, including PKM2, HK2, and GLUT1, exhibited significant downregulation following ASPSCR1 knockdown (Figure 4C) . These findings further affirm the regulatory function of ASPSCR1 in the glycolysis pathway, thereby strengthening our understanding of its role in OS. 3.5 ASPSCR1 regulates OS progression in a glycolysis-dependent manner PKM2-IN-3, an inhibitor of the PKM2 kinase, exerts its influence on cellular metabolism by inhibiting PKM2-mediated glycolysis. Our study observed a particularly intriguing phenomenon when ASPSCR1-overexpressing HOS and U2OS cells were treated with PKM2-IN-3. The enhanced glycolytic activity induced by ASPSCR1 overexpression was effectively reversed upon treatment with the PKM2-IN-3, as evidenced by the restoration of glucose levels, lactic acid production, ATP content (Figure 4D) , and the expression of key enzymes (Figure 4E) . Similarly, a comparable phenomenon was observed in terms of cell proliferation (Figure 4F) clone formation (Figure 4G) . This discovery underscores the pivotal role of glycolysis in ASPSCR1-mediated cellular functions and offers a novel strategic perspective for the treatment of OS. In summary, our research data robustly support the notion that ASPSCR1 regulates OS progression through the upregulation of glycolysis. Consequently, the combined targeted inhibition of ASPSCR1 and glycolysis emerges as a promising therapeutic strategy, as it has the potential to weaken the glycolytic pathway and thereby inhibit the growth of OS cells. 3.6 In vivo validation of the effect of ASPSCR1 on the progression of OS cells To comprehensively evaluate the specific role of ASPSCR1 in the progression of OS, we designed and conducted in vivo experiments. A xenograft tumor model was successfully established by subcutaneous injection of ASPSCR1 knockdown and control OS cells (HOS) into nude mice. During the subsequent observation period, we meticulously documented the growth of the tumors and monitored changes in tumor volume. Notably, the tumor volume formed by ASPSCR1 knockdown tumor cells in nude mice was significantly reduced (Figure 5A, B) . This reduction was further confirmed by a comparison of tumor weights after resection (Figure 5C) . To validate the knockdown effect of ASPSCR1, we extracted samples from various tumor tissues for Western blot analysis. The results unequivocally demonstrated the downregulation of ASPSCR1 expression in the ASPSCR1 knockdown group (Figure 5D) . Additionally, IHC staining analysis of tumor tissue sections revealed that ASPSCR1 knockdown not only decreased glycolytic activity, evidenced by reduced GLUT1 expression (Figure 5E, F) , but also inhibited tumor cell proliferation, as indicated by decreased Ki67 expression (Figure 5G) . In summary, our in vivo experiments provide compelling evidence supporting the crucial role of ASPSCR1 in promoting the progression of OS cells. 4. Discussion This study delineates a previously unrecognized oncogenic axis in OS progression, centering on ASPSCR1-mediated metabolic reprogramming. Our findings reveal three pivotal advances: (1) ASPSCR1 serves as a novel prognostic biomarker and therapeutic target in OS; (2) ASPSCR1 orchestrates OS by increasing glycolytic flux; (3) The ASPSCR1-glycolysis axis represents a metabolic vulnerability similar to canonical Warburg effect regulators. These discoveries fundamentally expand our understanding of OS pathobiology while addressing critical gaps in cancer metabolism research. While ASPSCR1 was previously characterized as a TFE3 fusion partner in alveolar soft part sarcoma (ASPS) 15 , our work uncovers its non-fusion oncogenic role in OS—a mechanistic divergence from prior reports. This finding challenges the paradigm that ASPSCR1 requires chromosomal translocation to exert oncogenic effects. Although earlier studies implicated metabolic reprogramming in OS progression 16 , our identification of ASPSCR1 as a glycolytic master regulator similar to Warburg effect mediators such as HIF-1α 17 or c-MYC 18 , ASPSCR1 appears to regulate glycolysis at multiple levels by controlling the expression of most glycolysis-related genes. Notably, ASPSCR1 displays distinct expression patterns and physiological functions compared to HIF-1α and c-myc. While HIF-1α and c-myc are well-known for their pro-tumorigenic activities, they also play essential physiological roles 19-21 . In contrast, ASPSCR1 primarily regulates GLUT4 translocation and glucose uptake in muscle and adipose tissues, although these functions are not exclusively dependent on ASPSCR1 9, 22 . Therefore, inhibiting ASPSCR1 can suppress glycolysis at multiple levels, while potentially causing fewer toxic side effects. Additionally, our cohort analysis revealing ASPSCR1's correlation with age/gender (Table 2/3) suggests demographic-specific therapeutic vulnerabilities, contrasting with pan-cancer metabolic targets like GLUT1 23-25 . The PKM2 inhibitor-mediated reversal of ASPSCR1-driven phenotypes highlights a synthetic lethal interaction, providing rationale for combinatory targeting—a strategy distinct from single-agent approaches in current trials. However, three limitations merit consideration: (1) The clinical cohort size (n=33) warrants validation in multicenter trials; (2) In vivo models using HOS cells may not fully recapitulate OS biology; (3) The precise mechanisms by which ASPSCR1 regulates the expression of glycolysis-related genes remain to be further elucidated. Future studies should explore ASPSCR1's nuclear-cytoplasmic shuttling mechanisms and investigate whether its metabolic role extends to other TFE3-related malignancies. This study repositions ASPSCR1 from a fusion partner to a central metabolic regulator. Our mechanistic dissection of the ASPSCR1-glycolysis axis unveils a preclinical targetable vulnerability that may revolutionize OS treatment paradigms, particularly for patients resistant to conventional therapies. These findings underscore the importance of context-specific metabolic mapping in precision oncology. Declarations Acknowledgements None. Disclosure: Funding Information This study was supported by Project for Constructing Achievements of Interdisciplinary Collaborative Innovation in the New Round of "Double First-Class" Discipline Construction in Heilongjiang Province of China (Grant No. 15041230003) and Heilongjiang Provincial Natural Science Foundation of China (Grant No. PL2024H217). Conflict of Interest The authors have no conflict of interest.” Ethics approval and consent to participate - Approval of the research protocol by an Institutional Reviewer Board: Written informed consent was obtained from all subjects, and human tissue collection was approved by the Ethics Committee of the First Affiliated Hospital, Heilongjiang University of Chinese Medicine (Approval number: HZYLLKT202341601). - Informed Consent: Not applicable. - Registry and the Registration No. of the study/trial: Not applicable. - Animal Studies: Not applicable. Patient consent for publication Not applicable. Authors' contributions Yang Liu, FeiWang and Geqiang Wang conceptualized and designed the study; Hanbing Song, Linqin He, Shuying Wu, and Youyou Li are responsible for the experimental operation; Hanbing Song, FeiWang, Linqin He, Shuying Wu, Youyou Li, Qian Zhang, and Geqiang Wang complete the data analysis; Hanbing Song, Shuying Wu, Youyou Li, Qian Zhang, Weixin Cai and Yang Liu jointly complete the article writing; Hanbing Song, FeiWang, and Yang Liu are responsible for article inspection. References Estrada-Villaseñor E, Pichardo-Bahena R, Cedeño-Garcidueñas AL, Delgado-Cedillo EA, Marín-Arriaga N, Arguelles-Pérez DA. Parosteal osteosarcoma with low grade chondrosarcoma and liposarcoma components. A rare histologic variant. Case report and literature review. Acta Ortop Mex . 2024; 38: 113-118. Wittig JC, Bickels J, Priebat D, et al. Osteosarcoma: a multidisciplinary approach to diagnosis and treatment. Am Fam Physician . 2002; 65: 1123-1132. Li S, Zhang H, Liu J, Shang G. 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Reprogramming of glucose metabolism: Metabolic alterations in the progression of osteosarcoma. J Bone Oncol . 2024; 44: 100521. Yang HL, Chang CW, Vadivalagan C, et al. Coenzyme Q(0) inhibited the NLRP3 inflammasome, metastasis/EMT, and Warburg effect by suppressing hypoxia-induced HIF-1α expression in HNSCC cells. Int J Biol Sci . 2024; 20: 2790-2813. Shen S, Yao T, Xu Y, Zhang D, Fan S, Ma J. CircECE1 activates energy metabolism in osteosarcoma by stabilizing c-Myc. Mol Cancer . 2020; 19: 151. Kumar H, Choi DK. Hypoxia Inducible Factor Pathway and Physiological Adaptation: A Cell Survival Pathway? Mediators Inflamm . 2015; 2015: 584758. Baena E, Gandarillas A, Vallespinós M, et al. c-Myc regulates cell size and ploidy but is not essential for postnatal proliferation in liver. Proc Natl Acad Sci U S A . 2005; 102: 7286-7291. Machi JF, Altilio I, Qi Y, et al. Endothelial c-Myc knockout disrupts metabolic homeostasis and triggers the development of obesity. Front Cell Dev Biol . 2024; 12: 1407097. Bogan JS. Ubiquitin-like processing of TUG proteins as a mechanism to regulate glucose uptake and energy metabolism in fat and muscle. Front Endocrinol (Lausanne) . 2022; 13: 1019405. Li F, He C, Yao H, et al. GLUT1 Regulates the Tumor Immune Microenvironment and Promotes Tumor Metastasis in Pancreatic Adenocarcinoma via ncRNA-mediated Network. J Cancer . 2022; 13: 2540-2558. Liu XS, Gao Y, Wu LB, et al. Comprehensive Analysis of GLUT1 Immune Infiltrates and ceRNA Network in Human Esophageal Carcinoma. Front Oncol . 2021; 11: 665388. You M, Jin J, Liu Q, Xu Q, Shi J, Hou Y. PPARα Promotes Cancer Cell Glut1 Transcription Repression. J Cell Biochem . 2017; 118: 1556-1562. Tables Table 1. Expression patterns in OS tissues and normal tissues revealed in immunohistochemistry analysis ASPSCR1 expression Tumor tissue Paracancerous tissues P value Cases Percentage Cases Percentage Low 38 52.80% 8 100.00% 0.044 High 34 47.20% 0 0.00% Table 2. Relationship between ASPSCR1 expression and tumor characteristics in patients with OS Features No. of patients ASPSCR1 expression P value low high All patients 72 38 34 Age 0.034 ≤30years 37 15 22 >30years 35 23 12 Gender 0.005 Male 48 31 17 Female 24 7 17 Table 3. Relationship between ASPSCR1 expression and tumor characteristics in patients with OS Tumor characteristics Index ASPSCR1 Gender Spearman correlation 0.334 Significance (two tailed) 0.004 N 72 Age Spearman correlation -0.252 Significance (two tailed) 0.003 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 13 Mar, 2026 Reviewers agreed at journal 11 Mar, 2026 Reviewers invited by journal 05 Mar, 2026 Editor assigned by journal 26 Dec, 2025 Submission checks completed at journal 26 Dec, 2025 First submitted to journal 25 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8446485","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":602136291,"identity":"2151d4d8-f0c8-47ea-a366-040ef5aa7b20","order_by":0,"name":"Hanbing Song","email":"","orcid":"","institution":"First Affiliated Hospital of Heilongjiang University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hanbing","middleName":"","lastName":"Song","suffix":""},{"id":602136292,"identity":"92fd18b6-116f-495e-bbda-86354395d913","order_by":1,"name":"Fei Wang","email":"","orcid":"","institution":"First Affiliated Hospital of Heilongjiang University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Wang","suffix":""},{"id":602136293,"identity":"984d31ff-f280-4ddb-86d3-fe3c41b7480e","order_by":2,"name":"Linqin He","email":"","orcid":"","institution":"First Affiliated Hospital of Heilongjiang University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Linqin","middleName":"","lastName":"He","suffix":""},{"id":602136294,"identity":"7b5133ed-40ad-4db0-b97d-2a0d30a200ee","order_by":3,"name":"Shuying Wu","email":"","orcid":"","institution":"Heilongjiang University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shuying","middleName":"","lastName":"Wu","suffix":""},{"id":602136295,"identity":"4a7927a8-be3d-4bc2-b500-6f67b4de4d6d","order_by":4,"name":"Youyou Li","email":"","orcid":"","institution":"Heilongjiang University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Youyou","middleName":"","lastName":"Li","suffix":""},{"id":602136296,"identity":"acf990ac-815b-475b-9e77-e30375241a7c","order_by":5,"name":"Wenqi Zhang","email":"","orcid":"","institution":"First Affiliated Hospital of Heilongjiang University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wenqi","middleName":"","lastName":"Zhang","suffix":""},{"id":602136297,"identity":"de3e5583-8642-4678-9f21-86117a2ade29","order_by":6,"name":"Qian Zhang","email":"","orcid":"","institution":"First Affiliated Hospital of Heilongjiang University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Zhang","suffix":""},{"id":602136298,"identity":"85b0b356-f5cc-4d02-bb92-a4cf6ba23860","order_by":7,"name":"Geqiang Wang","email":"","orcid":"","institution":"First Affiliated Hospital of Heilongjiang University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Geqiang","middleName":"","lastName":"Wang","suffix":""},{"id":602136299,"identity":"f7a7a7fc-7951-496d-82b5-2a46a8afe010","order_by":8,"name":"Weixin Cai","email":"","orcid":"","institution":"First Affiliated Hospital of Heilongjiang University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Weixin","middleName":"","lastName":"Cai","suffix":""},{"id":602136300,"identity":"88eaf48c-f57d-4d51-a9c0-e6455fb9b6fc","order_by":9,"name":"Yang Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYBACPgbGxgcfeGzq++UPH//woUJCTp6QFjYG5mbDGTJpjDNnsKUxzjhjYWzYQFALe5s0j80hxg03eNSYedsqEhkOENIikQi0JecAs8HtHraHM+dJJDA2MD98dAO/FqBfztxhk7xz9rjBx20SeewMbMbGOYRsmdnzjIfvQF6C5MxtEsWMDTxs0gS0tEnz/jsswXAgx0Cad45EYsMBYrTw8Bw2ELiRYybN20CMFp6HQO/zpCVI9hxLNpxxTMLYsJmAX/jZ0x+CojKBn7354IMPNXVy8uzNDx/j08IgkIAuwoxPOdiaA4RUjIJRMApGwYgHAHPmUsAPZAo+AAAAAElFTkSuQmCC","orcid":"","institution":"First Affiliated Hospital of Heilongjiang University of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Yang","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-12-25 05:38:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8446485/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8446485/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104445143,"identity":"553d65b2-f3f0-48c0-8e93-8e4478f325e2","added_by":"auto","created_at":"2026-03-11 19:52:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11512812,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eASPSCR1 is highly expressed in OS and is associated with poor prognosis. \u003c/strong\u003e(A) High expression of ASPSCR1 in OS suggests poor overall survival. (B) plots show the HRs of different factors calculate from multivariable Cox regression analysis. (C) Correlation of ASPSCR1 expression in osteosarcoma with other factors. (D) Representative image of IHC and HE staining of ASPSCR1 in OS tissue microarray. Magnification, ×200 and ×400.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8446485/v1/2ded31d961e0c740f17ca2f3.png"},{"id":104445144,"identity":"302dcbaa-9717-4a78-b62f-ae0094a459fe","added_by":"auto","created_at":"2026-03-11 19:52:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12961579,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe construction of a cell model with ASPSCR1 knockdown.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) RT-qPCR detected the expression of ASPSCR1 in different human OS cell lines（U2OS, SW-1353, HOS, 143）,*P\u0026lt;0.05**P\u0026lt;0.05,***P\u0026lt;0.05.(B) The expression of ASPSCR1 in U2OS cells was detected by RT-qPCR after cell transfection, n=3,*** P\u0026lt;0.001.(C) Western blot analyses of ASPSCR1-related proteins with or without ASPSCR1 knockdown（shASPACR1-1/2/3） in U2OS cells. GAPDH was used as the loading control n=3. (D) Representative images of GFP assays of OS cells (HOS、U2OS) transfected with shCtrl or shASPSCR1 (shASPSCR1-1 and shASPSCR1-3) . Scale bar, 50 μm. (E-F) The mRNA (E) and protein (F) levels of ASPSCR1 in U2OS cells and HOS cells after transfection with shASPSCR1 (shASPSCR1-1 and shASPSCR1-3) or shCtrl (control). ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8446485/v1/f0aef8052fc64c222524237d.png"},{"id":104445146,"identity":"0f38a391-7d17-46f6-81cd-814fb740f42a","added_by":"auto","created_at":"2026-03-11 19:52:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8437112,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of ASPSCR1 inhibits the progression of OS cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) CCK8 assay performed on HOS and U2OS cells after transfection to determine the cell viability. The proliferation speed of the shASPSCR1-1 and shASPSCR1-3 cells significantly reduced. **P\u0026lt;0.01, fold change of group shASPSCR1-1=-1.4, fold change of group shASPSCR1-3=-1.7. ***P\u0026lt;0.001, fold change of group shASPSCR1-1=-1.4, fold change of group shASPSCR1-3=-1.7. (B) Clone formation assay detected the HOS and U2OS cell reproductive capacity and the number of clones has significantly decreased in the shASPSCR1-1 and shASPSCR1-3 cells, **P\u0026lt;0.01. (C) The apoptosis rate of HOS and U2OS cells was detected by flow cytometry and was significantly increased in the shASPSCR1-1 and shASPSCR1-3 cells, *P\u0026lt;0.05**P\u0026lt;0.05.(D) Wound healing assays were performed in HOS and U2OS cells, **P\u0026lt;0.01***P\u0026lt;0.01. (E) Transwell assays were conducted to assess cell migration after knockdown of ASPSCR1, respectively, in HOS and U2OS cells compared with corresponding vector cells, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8446485/v1/c5afc6bae8fe72b5d82fc132.png"},{"id":104445147,"identity":"fb88a6a4-8f82-4a24-9c22-082565866867","added_by":"auto","created_at":"2026-03-11 19:52:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":14962525,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eASPSCR1 regulates OS progression through PKM2 mediated glycolysis pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)GSEA indicating significant correlations between ASPSCR1 mRNA expression and glycolysis pathway. (B) ATP levels, glucose consumption and lactic acid production in HOS/shASPSCR1 cells or U2OS/shASPSCR1 cells. Three independent experiments were performed. *p \u0026lt; 0.05 versus control. *P\u0026lt;0.05**P\u0026lt;0.01***P\u0026lt;0.001. (C) Western blot analysis of PKM2, HK2, GLUT1, ADH4, ALDOC, ALDOA, G6PD, FBP1, ADH6 and LDHA, in HOS-shASPSCR1 cells and U2OS-shASPSCR1 cells. (D) ATP levels, glucose consumption and lactic acid production in HOS and U2OS cells stably transfected with ASPSCR1 in the presence or absence of PKM2-IN-3. *p\u0026lt;0.05,**p \u0026lt; 0.01. (E) Western blotting and protein expression level of ASPSCR1 in HOS and U2OS cells stably transfected with ASPSCR1 in the presence or absence of PKM2-IN-3. Western blot analysis of PKM2, HK2, LDHA, ALDOC and β-Actin, in HOS and U2OS cells transfected with ASPSCR1 in the presence or absence of PKM2-IN-3.(F) CCK- 8 assays showing proliferation capacity of HOS(left) and U2OS(right) cells stably transfected with ASPSCR1 in the presence or absence of PKM2-IN-3. **p \u0026lt; 0.01，fold change=-1.4(left)，fold change=-1.6(right).***p \u0026lt; 0.01，fold change=-2.3(left)，fold change=-2.5(right)(G) Representative images and reproductive capacity of colony formation assays of OS cells transfected with ASPSCR1 in the presence or absence of PKM2-IN-3. Compared with the NC group, the number of clones in the ASRSCR1 group increased, and compared with the ASRSCR1 group, the number of clones in the ASRSCR1+PKM2-IN-3 group decreased. *p\u0026lt;0.05，**p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8446485/v1/8dbf54595cb2b30a13889d8b.png"},{"id":104445148,"identity":"a5cd1d6b-59a6-40c3-82d3-cf6deddfa8df","added_by":"auto","created_at":"2026-03-11 19:52:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":35627160,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo validation of the effect of ASPSCR1 on the progression of OS cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-C) Subc. inj. of sh-ASPSCR1 OS cells \u0026amp; ctrl. HOS cells into nude mice were done, and the xenograft tumor model was estab. successfully. During the subseq. obs. period, the tumor growth was rec. meticulously, and the dynamic changes were mon. continuously (n = 6), ***P\u0026lt;0.001.(D) The ASPSCR1 protein level of the HOS cells was detected by using Western blotting. (E-G) IHC staining of ASPSCR1, GLUT1 and Ki67 (scale bar, 50 μm) in tumor sections (n = 3 for each group).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8446485/v1/c46b78bf41571db297a75b07.png"},{"id":104808509,"identity":"798464e9-4a90-4fb5-911e-c01258edf46e","added_by":"auto","created_at":"2026-03-17 12:38:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":92781038,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8446485/v1/87e1cf68-503e-4f91-a592-86f2f70bb9a2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"ASPSCR1 promotes osteosarcoma progression through the upregulation of glycolysis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOsteosarcoma (OS), a highly malignant connective tissue tumor, ranks significantly in primary bone malignancies, posing severe threats to patients\u0026apos; lives and well-being. Characterized by tumor cells directly producing bone and osteoid tissues, it exhibits high malignancy, poor prognosis, and rapid progression with a tendency to metastasize to the lungs and other distant sites \u003csup\u003e1\u003c/sup\u003e. Current treatments primarily involve surgical resection combined with chemotherapy and radiotherapy \u003csup\u003e2\u003c/sup\u003e. In recent years, targeted therapies have shown remarkable progress, offering new hope for improving patient outcomes \u003csup\u003e3\u003c/sup\u003e. However, challenges such as limited drug options, high costs, and drug resistance persist \u003csup\u003e4\u003c/sup\u003e. Therefore, exploring the mechanisms underlying OS development is crucial for developing novel and effective targeted therapies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGlycolysis is a crucial metabolic pathway for tumor cells to acquire energy, particularly in hypoxic or rapidly proliferating tumor environments \u003csup\u003e5\u003c/sup\u003e. This metabolic reprogramming not only provides essential energy and biosynthetic precursors for tumor cells but also alters the tumor microenvironment, promoting invasion and metastasis\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e6\u003c/sup\u003e. In OS, the rapid growth and uncontrolled characteristics of tumor cells make them highly dependent on glucose uptake and glycolysis \u003csup\u003e7\u003c/sup\u003e. Therefore, developing targeted therapeutic strategies for OS from the perspective of glycolysis is of great theoretical and practical significance.\u003c/p\u003e\n\u003cp\u003eASPSCR1, fullname is \u0026apos;ASPSCR1 tether for SLC2A4, UBX domain containing\u0026apos;, also kown \u0026nbsp; as TUG, which sequesters the GLUT4 in intracellular vesicles in muscle and fat cells in the absence of insulin, and redistributes the GLUT4 to the plasma membrane within minutes of insulin stimulation \u003csup\u003e8, 9\u003c/sup\u003e. It shuttles between the nucleus and cytoplasm, regulating gene transcription and protein synthesis \u003csup\u003e10\u003c/sup\u003e. Notably, the ASPSCR1::TFE3 fusion protein, formed by the fusion of ASPSCR1 with TFE3, leverages TFE3\u0026apos;s basic helix-loop-helix (bHLH) domain to drive oncogenic transcription. This fusion protein regulates genes involved in lysosomal function, autophagy, and angiogenesis, serving as a central genetic driver in alveolar soft part sarcoma (ASPS) and Xp11 translocation renal cell carcinoma (RCC) \u003csup\u003e11\u003c/sup\u003e. Research indicates that ASPSCR1::TFE3 preferentially binds to super-enhancers, promoting angiogenesis and supporting tumor growth. In TFE3-rearranged RCC, patients with ASPSCR1-TFE3 fusion exhibit better responses to immunotherapy combinations\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e12\u003c/sup\u003e. ASPSCR1\u0026apos;s somatic variations are also linked to poor prognosis in hereditary diffuse gastric cancer (HDGC) \u003csup\u003e13\u003c/sup\u003e. Moreover, ASPSCR1 mitigates oxidative stress, proliferation, and migration of cholangiocarcinoma cells induced by rCsNOSIP from Opisthorchis felineus\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e14\u003c/sup\u003e. These findings highlight ASPSCR1\u0026apos;s complex roles across different cancer contexts.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, we aim to comprehensively explore ASPSCR1\u0026apos;s role and mechanisms in OS. We will analyze and validate ASPSCR1 expression in OS tissues and cell lines, knock down ASPSCR1 expression, and investigate its effects on cell proliferation, migration, invasion, and glycolysis. Our goal is to uncover ASPSCR1\u0026apos;s functions and mechanisms in OS, particularly focusing on its downstream mechanism in glycolysis regulation. This research will provide new insights into OS pathogenesis and may identify novel therapeutic targets for treatment.\u0026nbsp;\u003c/p\u003e"},{"header":"2.\tMaterials and methods","content":"\u003ch2\u003e2.1 Tissue Samples\u003c/h2\u003e\n\u003cp\u003eOS tissue sections (n=80, model: LBN802) were provided by Xi\u0026apos;an Tebos Pharmaceutical Technology Co., Ltd. Additionally, normal bone tissue sections (n=9, model: YBRHost09-M002) were obtained from Shanghai YiBeiRui Bioscience Co., Ltd. The tumor samples were collected from patients with primary OS who underwent surgical resection. All samples were evaluated by pathologists andparaffin-embedded. Patient data included sex, age, and other clinical parameters. Written informed consent was obtained from all subjects, and human tissue collection was approved by the Ethics Committee of the First Affiliated Hospital, Heilongjiang University of Chinese Medicine (Approval number: HZYLLKT202341601).\u003c/p\u003e\n\u003ch2\u003e2.2 Immunohistochemistry (IHC) and Hematoxylin-Eosin (HE) staining\u003c/h2\u003e\n\u003cp\u003eParaffin-embedded tissue samples sections were subjected to IHC analysis for ASPSCR1, Ki67, and GLUT1 proteins. Tissue sections were heated at 60\u0026deg;C for 2 hours, deparaffinized with xylene, and rehydrated through a descending ethanol series. Sections were then incubated with primary antibodies against ASPSCR1 (PhD Bio, 1:100, M05168), Ki67 (Abcam, 1:200, ab16667), and GLUT1 (PhD Bio, 1:100, BA3481-2) at 4\u0026deg;C for 12 hours. The following day, sections were washed with PBS and incubated with a goat anti-rabbit IgG H\u0026amp;L (HRP) pre-adsorbed secondary antibody (Abcam, 1:200, ab97080) at room temperature for 2 hours. Staining was visualized using the ImmunoPure Metal-Enhanced DIY ELISA Builder (DAB) substrate kit according to the manufacturer\u0026apos;s instructions, and results were observed under a light microscope. Scoring of staining intensity and the percentage of positive cells was performed by two pathologists. The IHC score was calculated as the sum of the intensity and percentage scores. Tumor tissue sections were also subjected to HE staining. Sections were fixed in 95% ethanol, washed, stained with hematoxylin for nuclear staining, followed by eosin for cytoplasmic staining, and then air-dried.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e2.3 Cell lines and Cultures\u003c/h2\u003e\n\u003cp\u003eHuman OS cell lines (U2OS, SW-1353, HOS, 143B) and normal human osteoblasts (Chondrocytes) were obtained from the Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). OS cell lines were cultured in Dulbecco\u0026apos;s Modified Eagle Medium (DMEM, Biological Industries, Israel) supplemented with 10% fetal bovine serum (FBS, Ausbian VS500T), 1% streptomycin, and 1% penicillin. Chondrocyte cells were cultured in DMEM/F12. All cells were maintained at 37\u0026deg;C with 5% CO₂ in a humidified incubator.All cell lines used in this study were authenticated via STR profiling and verified as mycoplasma-free.\u003c/p\u003e\n\u003ch2\u003e2.4 Lentivirus Production and Infection (RNAi)\u003c/h2\u003e\n\u003cp\u003eLentiviral vectors containing shRNA sequences targeting ASPSCR1 and negative control shRNA were obtained from Shanghai YiBeiRui BioPharmaceuticals Co., Ltd. U2OS and HOS cells were seeded in 6-well plates at 2\u0026times;10⁵ cells per well. After 24 hours, cells were infected with lentivirus following the manufacturer\u0026apos;s instructions. Following a 72-hour incubation, cells were treated with fresh medium containing 10 \u0026micro;g/mL puromycin for selection. The sequences of used in this study are listed as follows: shASPSCR1-1: 5\u0026apos;-CCTGCACCTAAGTCTGAGCCA-3\u0026apos;; shASPSCR1-2: 5\u0026apos;-GCCTGATGAGTTCTTTGAGCT-3\u0026apos;; shASPSCR1-3: 5\u0026apos;-TCGAGGTTGCAGGACTCTTTC-3\u0026apos;.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e2.5 Quantitative real-time Polymerase Chain Reaction (qPCR)\u003c/h2\u003e\n\u003cp\u003eTotal RNA was isolated from OS tissues and cells using TRIzol reagent (Sigma, T9424-100mL) according to the manufacturer\u0026apos;s instructions. cDNA was synthesized using the HiScript QRT SuperMix Kit (Vazyme, R123-01) and stored at -80\u0026deg;C. The following primer pairs were used: GAPDH forward, 5\u0026apos;-TGACTTCAACAGCGACACCCA-3\u0026apos;; GAPDH reverse, 5\u0026apos;-CACCCTGTTGCTGTAGCCAAA-3\u0026apos;; ASPSCR1 forward, 5\u0026apos;-AACATGGTTCGCATCGCTTTG-3\u0026apos;; ASPSCR1 reverse, 5\u0026apos;-ACCCGTCACCTCATCCCTC-3\u0026apos;.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e2.6 Western Blot (WB)\u003c/h2\u003e\n\u003cp\u003eCells were washed once with cold phosphate-buffered saline (PBS) and lysed in RIPA lysis buffer for 10 minutes. They were then centrifuged at 12,000 rpm for 10 minutes at 4\u0026deg;C to obtain the supernatant. Equal amounts of protein were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Millipore, USA). The membranes were blocked using 5% skimmed milk for 2 hours and then incubated with primary antibodies overnight at 4\u0026deg;C. The primary antibodies used were anti-ASPSCR1 (1:1000, BOSTER), GAPDH (1:30,000, Proteintech), PKM2 (1:5000, Proteintech), HK2 (1:3000, Proteintech), GLUT1 (1:1000, Proteintech), ADH4 (1:2000, Abcam), ALDOC (1:5000, Proteintech), ALDOA (1:3000, Proteintech), G6PD (1:1000, Abcam), FBP1 (1:2000, Proteintech), ADH6 (1:5000, Proteintech), LDHA (1:3000, Abcam), and \u0026beta;-Actin (1:4000, Proteintech). After incubation with appropriate anti-mouse/rabbit secondary antibodies for 1 hour at room temperature, immunoreactive bands were detected using a chemiluminescence kit (Millipore, USA).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e2.7 Cell Proliferation Assay\u003c/h2\u003e\n\u003cp\u003eCell proliferation was assessed using the Cell Counting Kit-8 (Sigma, 96992). U2OS and HOS cells (1 \u0026times; 10\u0026sup3; cells/well) were seeded in 96-well plates (Corning, 3596) and cultured for 24, 48, and 72 hours. Each well received 10 \u0026mu;L of CCK-8 solution, followed by incubation at 37\u0026deg;C with 5% CO₂ for 2 hours. Absorbance at 450 nm was measured using a microplate reader (Tecan Infinite, M2009PR).\u003c/p\u003e\n\u003ch2\u003e2.8 Colony Formation Assay\u003c/h2\u003e\n\u003cp\u003eU2OS and HOS cells with ASPSCR1 knockdown or overexpression were seeded in 6-well plates at 500 cells per well 5 days post-infection. Cells were cultured for 8 days with medium changes every 3 days. Cell colonies were photographed under a fluorescence microscope (Olympus, IX71), fixed with 4% paraformaldehyde (Sinopharm Chemical Reagent Co., Ltd., AR Shanghai Test) for 30 minutes, and stained with Giemsa solution (Shanghai Dingguo Biotechnology Co., Ltd., AR-0752) for 30 minutes. Colonies were counted after air-drying and washing with D-Hanks.\u003c/p\u003e\n\u003ch2\u003e2.9 Flow Cytometry\u003c/h2\u003e\n\u003cp\u003eApoptosis was assessed using the Annexin V-FITC/Propidium Iodide (PI) Apoptosis Detection Kit (eBioscience, 88-8007-7). Transfected cells were trypsinized, collected, and washed twice with cold 1\u0026times; PBS. Cells were suspended in 400 \u0026mu;L 1\u0026times; binding buffer, stained with 5 \u0026mu;L Annexin V-FITC for 15 minutes at 4\u0026deg;C in the dark, followed by 10 \u0026mu;L PI for 10 minutes. Stained cells were analyzed by flow cytometry to determine early and late apoptosis.\u003c/p\u003e\n\u003ch2\u003e2.10 Scratch Assay\u003c/h2\u003e\n\u003cp\u003eU2OS and HOS cells were seeded in 6-well plates with serum-enriched medium (Corning, 10-013-CVR). Upon reaching over 90% confluence, a scratch was made using a scratch tool (VP Scientific, VP408FH). After washing with D-Hanks, cells were cultured in fresh DMEM (Corning, 10-013-CVR) with 2% FBS. Scratch area changes were observed under a microscope (Olympus, IX73) at 0 h and 24 h. Cellomics (Thermo, ArrayScan VT1) quantified the migrated area.\u003c/p\u003e\n\u003ch2\u003e2.11 Transwell Assay\u003c/h2\u003e\n\u003cp\u003eCell invasion was assessed using Transwell plates (Corning, 3422). Logarithmic growth phase cells were resuspended in serum-free medium and seeded in the upper chamber coated with Matrigel. The lower chamber contained 600 \u0026mu;L of complete medium with 30% fetal bovine serum. After 24 hours of incubation, cells were stained and counted.\u003c/p\u003e\n\u003ch2\u003e2.12 Measurement of Glycolysis Levels (GLU, LA, ATP)\u003c/h2\u003e\n\u003cp\u003eThe levels of glucose (GLU), lactic acid (LA), and adenosine triphosphate (ATP) were measured to evaluate glycolysis activity. All assays were performed in triplicate and the results were normalized to protein concentration or cell number to ensure accuracy and reproducibility.\u003c/p\u003e\n\u003cp\u003eFor glucose consumption assay, GLU concentration was determined using the o-toluidine method with a glucose assay kit (Solarbio, Beijing, China, product number: BC2500, specification: 50T/48S). Cells were cultured in 96-well plates at a density of 5\u0026times;10\u0026sup3; cells per well and incubated at 37\u0026deg;C for 48 hours. The absorbance was measured at 505 nm to calculate glucose consumption levels.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor ATP content assay, ATP levels were measured using an ATP content assay kit (Solarbio, Beijing, China, product number: BC0300, specification: 50T/48S). Cells were seeded in 96-well plates at 5\u0026times;10\u0026sup3; cells per well. Cell extracts were prepared and analyzed according to the manufacturer\u0026apos;s instructions. The absorbance at 340 nm was measured to determine ATP content.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor LA production assay, LA production was measured using a LA content assay kit (Solarbio, Beijing, China, product number: BC2230, specification: 50T/24S). Cells were seeded in 96-well plates at 5\u0026times;10\u0026sup3; cells per well. Cell extracts were prepared and analyzed according to the manufacturer\u0026apos;s instructions. The absorbance at 570 nm was measured to determine LA content.\u003c/p\u003e\n\u003ch2\u003e2.13 Tumorigenicity Assay\u003c/h2\u003e\n\u003cp\u003eTwelve female BALB/c nude mice (4-6 weeks old) were purchased from Jiangsu Jicui Pharmachem Bio-technology Co. Ltd. The mice were housed under pathogen-free conditions. HOS cells (1\u0026times;10⁷ cells in 200 \u0026mu;L) were injected subcutaneously into the right flank of each mouse. The mice were randomly divided into two groups: negative control (NC) and knockdown (KD). Tumor growth was measured at specified time points (7, 11, 14, 18, and 21 days) using calipers, and tumor volume was calculated using the formula (\u0026pi;/6) \u0026times; L\u0026times; W\u0026sup2;, where L is the tumor length and W is the tumor width. Mice were euthanized 25 days post-injection, and tumor weights were measured. Tissues were stored at -80\u0026deg;C for subsequent analysis. All animal experiments were approved by the Ethics Committee of The First Affiliated Hospital of Heilongjiang University of Traditional Chinese Medicine (Approval number: HZYLLKT202341601) and conducted in accordance with their guidelines and rules.\u003c/p\u003e\n\u003ch2\u003e2.14 Statistical Methods\u003c/h2\u003e\n\u003cp\u003eAll data are presented as the mean \u0026plusmn; standard error of the mean (SEM) and were analyzed using GraphPad Prism 8.0 (La Jolla, USA). Differences between groups were assessed using Student\u0026apos;s t-test or one-way analysis of variance (ANOVA). All statistical tests were two-sided, and a p-value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"3.\tResults","content":"\u003ch2\u003e3.1 ASPSCR1 is highly expressed in OS and is associated with poor prognosis\u003c/h2\u003e\n\u003cp\u003eTo gain a comprehensive understanding of ASPSCR1\u0026apos;s role in OS, we analyzed data from the GEO database (GSE42352) and Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database. Our analysis revealed that ASPSCR1 expression levels were significantly higher in OS tissues than in normal tissues \u003cstrong\u003e(Figure 1A)\u003c/strong\u003e. We then categorized the samples based on the media2n expression value of ASPSCR1, dividing them into high-expression and low-expression groups. Kaplan-Meier survival analysis with the log-rank test demonstrated that patients with high ASPSCR1 expression had significantly shorter overall survival compared to those with low expression (hazard ratio (HR) = 2.88 (95% CI: 1.29-6.44), P = 0.00707) \u003cstrong\u003e(Figure 1B)\u003c/strong\u003e. A Cox multivariate regression analysis was performed to evaluate the association between the expression of ASPSCR1 and clinical characteristics. These findings suggest that the expression level of ASPSCR1 can serve as an independent prognostic factor (HR=2.62 (95% CI: 1.59-4.34), P = 0.000174), irrespective of other clinical indicators \u003cstrong\u003e(Figure 1C)\u003c/strong\u003e. To gain a profound understanding of the clinical significance of ASPSCR1 in OS, we constructed tissue microarrays using normal and tumor tissues from OS patients and performed HE staining and IHC analysis. By comparing the staining patterns and conducting quantitative analysis \u003cstrong\u003e(Table 1)\u003c/strong\u003e, we found that ASPSCR1 expression was significantly elevated in OS tissues compared to normal tissues \u003cstrong\u003e(Figure 1D)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo delve deeper into the relationship between ASPSCR1 expression levels and the clinicopathological characteristics of OS, we employed statistical methods for an in-depth analysis of the data. Initially, we classified samples into ASPSCR1 high-expression and low-expression groups, using the median expression value across all OS samples as the threshold. Subsequently, based on patients\u0026apos; clinical information, we utilized the Mann-Whitney U test to reveal a significant positive correlation between ASPSCR1 expression levels and both gender and age of the patients \u003cstrong\u003e(Table 2)\u003c/strong\u003e. To further validate this result, we conducted a Pearson rank correlation analysis, which again demonstrated that ASPSCR1 expression levels increased correspondingly with the advancement of tumor malignancy in patients \u003cstrong\u003e(Table 3)\u003c/strong\u003e. Our above statistical analyses further confirmed that ASPSCR1 expression levels are closely associated with tumor malignancy and patient outcomes, highlighting its importance in the clinical management of OS.\u003c/p\u003e\n\u003ch2\u003e3.2 The construction of ASPSCR1 knockdown in OS cells\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eTo elucidate the specific role of ASPSCR1 in the phenotypic traits and biological functions of OS cells, we meticulously designed and executed a comprehensive series of cellular-level experiments. Initially, we screened various OS cell lines for their ASPSCR1 expression levels using qPCR technology. Based on this screening, two cell lines, HOS and U2OS, which exhibited relatively high ASPSCR1 expression, were selected for further experimentation \u003cstrong\u003e(Figure 2A)\u003c/strong\u003e. To establish effective HOS and U2OS cell models, we identified suitable interference targets through a rigorous process involving qPCR and WB analyses \u003cstrong\u003e(Figure 2B, C)\u003c/strong\u003e. Guided by these screening results, we meticulously selected the two shASPSCR1 sequences that demonstrated the highest knockdown efficiency, with the aim of developing an OS cell model characterized by downregulated ASPSCR1 expression.\u003c/p\u003e\n\u003cp\u003eSubsequently, we underwent a series of meticulous construction and validation procedures. These included observing the infection efficiency under a green fluorescent protein microscope, which confirmed that the cell infection efficiency surpassed 80% and maintained good cell health \u003cstrong\u003e(Figure 2D)\u003c/strong\u003e. Furthermore, we conducted double validation of the knockdown efficiency through additional analyses \u003cstrong\u003e(Figure 2E, F)\u003c/strong\u003e. Following these rigorous steps, we successfully constructed HOS and U2OS cell models featuring dual-target knockdown of ASPSCR1.\u003c/p\u003e\n\u003ch2\u003e3.3 Knockdown of ASPSCR1 inhibits the progression of OS cells\u003c/h2\u003e\n\u003cp\u003eIn subsequent functional validation experiments, we employed the CCK8 Assay to observe that the knockdown of ASPSCR1 significantly impaired the proliferative capacity of HOS and U2OS cells (Figure 3A). Furthermore, ASPSCR1-knockdown cells exhibited a reduced clone formation ability, forming smaller and fewer clones compared to control cells (Figure 3B). These observations underscore the pivotal role of ASPSCR1 in facilitating cell growth. Additionally, flow cytometry analysis revealed that the reduction of ASPSCR1 expression augmented the apoptotic tendency of HOS and U2OS cells (Figure 3C), further emphasizing its crucial role in regulating cellular fate. To comprehensively assess the impact of ASPSCR1 on the migratory and invasive capabilities of OS cells, we conducted scratch and Transwell experiments. The results demonstrated that ASPSCR1 knockdown markedly inhibited the migration and invasion of OS cells (Figure 3D, E), providing compelling evidence for its potential role in the malignant progression of OS. In summary, our research findings not only unveiled the abnormally elevated expression of ASPSCR1 in OS cells but also validated its essential role in promoting cell proliferation, inhibiting apoptosis, and enhancing cellular migration and invasion abilities through a series of rigorous functional experiments.\u003c/p\u003e\n\u003ch2\u003e3.4 Knockdown of ASPSCR1 inhibits glycolysis levels in OS cells\u003c/h2\u003e\n\u003cp\u003eIn our investigation of the molecular mechanisms underlying OS, we discovered that the high expression of ASPSCR1 was significantly enriched in the glycan biosynthesis, suggesting its potential role in tumor glucose metabolism \u003cstrong\u003e(Figure 4A)\u003c/strong\u003e. To substantiate this observation, we conducted further experimental validations, which revealed that the knockdown of ASPSCR1 markedly impaired the glycolytic capacity of HOS and U2OS cells. This was evident from decreased glucose uptake, reduced ATP levels, and limited lactate production \u003cstrong\u003e(Figure 4B)\u003c/strong\u003e, all of which serve as direct indicators of diminished glycolysis activity. Concurrently, we observed that multiple crucial proteins involved in the glycolysis pathway, including PKM2, HK2, and GLUT1, exhibited significant downregulation following ASPSCR1 knockdown \u003cstrong\u003e(Figure 4C)\u003c/strong\u003e. These findings further affirm the regulatory function of ASPSCR1 in the glycolysis pathway, thereby strengthening our understanding of its role in OS.\u003c/p\u003e\n\u003ch2\u003e3.5 ASPSCR1 regulates OS progression in a glycolysis-dependent manner\u003c/h2\u003e\n\u003cp\u003ePKM2-IN-3, an inhibitor of the PKM2 kinase, exerts its influence on cellular metabolism by inhibiting PKM2-mediated glycolysis. Our study observed a particularly intriguing phenomenon when ASPSCR1-overexpressing HOS and U2OS cells were treated with PKM2-IN-3. The enhanced glycolytic activity induced by ASPSCR1 overexpression was effectively reversed upon treatment with the PKM2-IN-3, as evidenced by the restoration of glucose levels, lactic acid production, ATP content \u003cstrong\u003e(Figure 4D)\u003c/strong\u003e, and the expression of key enzymes \u003cstrong\u003e(Figure 4E)\u003c/strong\u003e. Similarly, a comparable phenomenon was observed in terms of cell proliferation \u003cstrong\u003e(Figure 4F)\u003c/strong\u003e clone formation \u003cstrong\u003e(Figure 4G)\u003c/strong\u003e. This discovery underscores the pivotal role of glycolysis in ASPSCR1-mediated cellular functions and offers a novel strategic perspective for the treatment of OS. In summary, our research data robustly support the notion that ASPSCR1 regulates OS progression through the upregulation of glycolysis. Consequently, the combined targeted inhibition of ASPSCR1 and glycolysis emerges as a promising therapeutic strategy, as it has the potential to weaken the glycolytic pathway and thereby inhibit the growth of OS cells.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e3.6 In vivo validation of the effect of ASPSCR1 on the progression of OS cells\u003c/h2\u003e\n\u003cp\u003eTo comprehensively evaluate the specific role of ASPSCR1 in the progression of OS, we designed and conducted in vivo experiments. A xenograft tumor model was successfully established by subcutaneous injection of ASPSCR1 knockdown and control OS cells (HOS) into nude mice. During the subsequent observation period, we meticulously documented the growth of the tumors and monitored changes in tumor volume. Notably, the tumor volume formed by ASPSCR1 knockdown tumor cells in nude mice was significantly reduced \u003cstrong\u003e(Figure 5A, B)\u003c/strong\u003e. This reduction was further confirmed by a comparison of tumor weights after resection \u003cstrong\u003e(Figure 5C)\u003c/strong\u003e. To validate the knockdown effect of ASPSCR1, we extracted samples from various tumor tissues for Western blot analysis. The results unequivocally demonstrated the downregulation of ASPSCR1 expression in the ASPSCR1 knockdown group \u003cstrong\u003e(Figure 5D)\u003c/strong\u003e. Additionally, IHC staining analysis of tumor tissue sections revealed that ASPSCR1 knockdown not only decreased glycolytic activity, evidenced by reduced GLUT1 expression \u003cstrong\u003e(Figure 5E, F)\u003c/strong\u003e, but also inhibited tumor cell proliferation, as indicated by decreased Ki67 expression \u003cstrong\u003e(Figure 5G)\u003c/strong\u003e. In summary, our in vivo experiments provide compelling evidence supporting the crucial role of ASPSCR1 in promoting the progression of OS cells.\u0026nbsp;\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study delineates a previously unrecognized oncogenic axis in OS progression, centering on ASPSCR1-mediated metabolic reprogramming. Our findings reveal three pivotal advances: (1) ASPSCR1 serves as a novel prognostic biomarker and therapeutic target in OS; (2) ASPSCR1 orchestrates OS by increasing glycolytic flux; (3) The ASPSCR1-glycolysis axis represents a metabolic vulnerability similar to canonical Warburg effect regulators. These discoveries fundamentally expand our understanding of OS pathobiology while addressing critical gaps in cancer metabolism research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhile ASPSCR1 was previously characterized as a TFE3 fusion partner in alveolar soft part sarcoma (ASPS) \u003csup\u003e15\u003c/sup\u003e, our work uncovers its non-fusion oncogenic role in OS\u0026mdash;a mechanistic divergence from prior reports. This finding challenges the paradigm that ASPSCR1 requires chromosomal translocation to exert oncogenic effects. Although earlier studies implicated metabolic reprogramming in OS progression\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e16\u003c/sup\u003e, our identification of ASPSCR1 as a glycolytic master regulator similar to Warburg effect mediators such as HIF-1\u0026alpha; \u003csup\u003e17\u003c/sup\u003e or c-MYC\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e18\u003c/sup\u003e, ASPSCR1 appears to regulate glycolysis at multiple levels by controlling the expression of most glycolysis-related genes. Notably, ASPSCR1 displays distinct expression patterns and physiological functions compared to HIF-1\u0026alpha; and c-myc. While HIF-1\u0026alpha; and c-myc are well-known for their pro-tumorigenic activities, they also play essential physiological roles \u003csup\u003e19-21\u003c/sup\u003e. In contrast, ASPSCR1 primarily regulates GLUT4 translocation and glucose uptake in muscle and adipose tissues, although these functions are not exclusively dependent on ASPSCR1 \u003csup\u003e9, 22\u003c/sup\u003e. Therefore, inhibiting ASPSCR1 can suppress glycolysis at multiple levels, while potentially causing fewer toxic side effects. \u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eAdditionally, our cohort analysis revealing ASPSCR1\u0026apos;s correlation with age/gender (Table 2/3) suggests demographic-specific therapeutic vulnerabilities, contrasting with pan-cancer metabolic targets like GLUT1 \u003csup\u003e23-25\u003c/sup\u003e. The PKM2 inhibitor-mediated reversal of ASPSCR1-driven phenotypes highlights a synthetic lethal interaction, providing rationale for combinatory targeting\u0026mdash;a strategy distinct from single-agent approaches in current trials.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHowever, three limitations merit consideration: (1) The clinical cohort size (n=33) warrants validation in multicenter trials; (2) In vivo models using HOS cells may not fully recapitulate OS biology; (3) The precise mechanisms by which ASPSCR1 regulates the expression of glycolysis-related genes remain to be further elucidated. Future studies should explore ASPSCR1\u0026apos;s nuclear-cytoplasmic shuttling mechanisms and investigate whether its metabolic role extends to other TFE3-related malignancies.\u003c/p\u003e\n\u003cp\u003eThis study repositions ASPSCR1 from a fusion partner to a central metabolic regulator. Our mechanistic dissection of the ASPSCR1-glycolysis axis unveils a preclinical targetable vulnerability that may revolutionize OS treatment paradigms, particularly for patients resistant to conventional therapies. These findings underscore the importance of context-specific metabolic mapping in precision oncology.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Information\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was supported by Project for Constructing Achievements of Interdisciplinary Collaborative Innovation in the New Round of \u0026quot;Double First-Class\u0026quot; Discipline Construction in Heilongjiang Province of China (Grant No. 15041230003) and Heilongjiang Provincial Natural Science Foundation of China (Grant No. PL2024H217).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflict of interest.\u0026rdquo;\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003e- Approval of the research protocol by an Institutional Reviewer Board: Written informed consent was obtained from all subjects, and human tissue collection was approved by the Ethics Committee of the First Affiliated Hospital, Heilongjiang University of Chinese Medicine (Approval number: HZYLLKT202341601).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;- Informed Consent: \u0026nbsp;Not applicable.\u003cbr\u003e\u0026nbsp; - Registry and the Registration No. of the study/trial: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;- Animal Studies: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYang Liu, FeiWang and Geqiang Wang conceptualized and designed the study; Hanbing Song, Linqin He, Shuying Wu, and Youyou Li are responsible for the experimental operation; Hanbing Song, FeiWang, Linqin He, Shuying Wu, Youyou Li, Qian Zhang, and Geqiang Wang complete the data analysis; Hanbing Song, Shuying Wu, Youyou Li, Qian Zhang, Weixin Cai and Yang Liu jointly complete the article writing; Hanbing Song, FeiWang, and Yang Liu are responsible for article inspection.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eEstrada-Villase\u0026ntilde;or E, Pichardo-Bahena R, Cede\u0026ntilde;o-Garcidue\u0026ntilde;as AL, Delgado-Cedillo EA, Mar\u0026iacute;n-Arriaga N, Arguelles-P\u0026eacute;rez DA. 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Gene expression profiling of alveolar soft-part sarcoma (ASPS). \u003cem\u003eBMC Cancer\u003c/em\u003e. 2009; 9: 22.\u003c/li\u003e\n\u003cli\u003ePozner A, Li L, Verma SP, et al. ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97. \u003cem\u003eNat Commun\u003c/em\u003e. 2024; 15: 1165.\u003c/li\u003e\n\u003cli\u003eZhao J, Tang Y, Hu X, et al. Patients with ASPSCR1-TFE3 fusion achieve better response to ICI based combination therapy among TFE3-rearranged renal cell carcinoma. \u003cem\u003eMol Cancer\u003c/em\u003e. 2024; 23: 132.\u003c/li\u003e\n\u003cli\u003eLiu ZX, Zhang XL, Zhao Q, et al. Whole-Exome Sequencing Among Chinese Patients With Hereditary Diffuse Gastric Cancer. \u003cem\u003eJAMA Netw Open\u003c/em\u003e. 2022; 5: e2245836.\u003c/li\u003e\n\u003cli\u003eBian M, Li S, Zhou H, et al. ASPSCR-1 and Sirt-5 alleviate Clonorchis liver fluke rCsNOSIP-induced oxidative stress, proliferation, and migration in cholangiocarcinoma cells. \u003cem\u003ePLoS Negl Trop Dis\u003c/em\u003e. 2023; 17: e0011727.\u003c/li\u003e\n\u003cli\u003eArgani P, Wobker SE, Gross JM, Matoso A, Fletcher CDM, Antonescu CR. PEComa-like Neoplasms Characterized by ASPSCR1-TFE3 Fusion: Another Face of TFE3-related Mesenchymal Neoplasia. \u003cem\u003eAm J Surg Pathol\u003c/em\u003e. 2022; 46: 1153-1159.\u003c/li\u003e\n\u003cli\u003eAn F, Chang W, Song J, et al. Reprogramming of glucose metabolism: Metabolic alterations in the progression of osteosarcoma. \u003cem\u003eJ Bone Oncol\u003c/em\u003e. 2024; 44: 100521.\u003c/li\u003e\n\u003cli\u003eYang HL, Chang CW, Vadivalagan C, et al. Coenzyme Q(0) inhibited the NLRP3 inflammasome, metastasis/EMT, and Warburg effect by suppressing hypoxia-induced HIF-1\u0026alpha; expression in HNSCC cells. \u003cem\u003eInt J Biol Sci\u003c/em\u003e. 2024; 20: 2790-2813.\u003c/li\u003e\n\u003cli\u003eShen S, Yao T, Xu Y, Zhang D, Fan S, Ma J. CircECE1 activates energy metabolism in osteosarcoma by stabilizing c-Myc. \u003cem\u003eMol Cancer\u003c/em\u003e. 2020; 19: 151.\u003c/li\u003e\n\u003cli\u003eKumar H, Choi DK. Hypoxia Inducible Factor Pathway and Physiological Adaptation: A Cell Survival Pathway? \u003cem\u003eMediators Inflamm\u003c/em\u003e. 2015; 2015: 584758.\u003c/li\u003e\n\u003cli\u003eBaena E, Gandarillas A, Vallespin\u0026oacute;s M, et al. c-Myc regulates cell size and ploidy but is not essential for postnatal proliferation in liver. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e. 2005; 102: 7286-7291.\u003c/li\u003e\n\u003cli\u003eMachi JF, Altilio I, Qi Y, et al. Endothelial c-Myc knockout disrupts metabolic homeostasis and triggers the development of obesity. \u003cem\u003eFront Cell Dev Biol\u003c/em\u003e. 2024; 12: 1407097.\u003c/li\u003e\n\u003cli\u003eBogan JS. Ubiquitin-like processing of TUG proteins as a mechanism to regulate glucose uptake and energy metabolism in fat and muscle. \u003cem\u003eFront Endocrinol (Lausanne)\u003c/em\u003e. 2022; 13: 1019405.\u003c/li\u003e\n\u003cli\u003eLi F, He C, Yao H, et al. GLUT1 Regulates the Tumor Immune Microenvironment and Promotes Tumor Metastasis in Pancreatic Adenocarcinoma via ncRNA-mediated Network. \u003cem\u003eJ Cancer\u003c/em\u003e. 2022; 13: 2540-2558.\u003c/li\u003e\n\u003cli\u003eLiu XS, Gao Y, Wu LB, et al. Comprehensive Analysis of GLUT1 Immune Infiltrates and ceRNA Network in Human Esophageal Carcinoma. \u003cem\u003eFront Oncol\u003c/em\u003e. 2021; 11: 665388.\u003c/li\u003e\n\u003cli\u003eYou M, Jin J, Liu Q, Xu Q, Shi J, Hou Y. PPAR\u0026alpha; Promotes Cancer Cell Glut1 Transcription Repression. \u003cem\u003eJ Cell Biochem\u003c/em\u003e. 2017; 118: 1556-1562.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"648\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\" style=\"width: 648px;\"\u003e\n \u003cp\u003eTable 1. Expression patterns in OS tissues and normal tissues revealed in immunohistochemistry analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 165px;\"\u003e\n \u003cp\u003eASPSCR1 expression\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 199px;\"\u003e\n \u003cp\u003eTumor tissue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 199px;\"\u003e\n \u003cp\u003eParacancerous tissues\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 84px;\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003eCases\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003ePercentage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003eCases\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003ePercentage\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 165px;\"\u003e\n \u003cp\u003eLow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e52.80%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e100.00%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 84px;\"\u003e\n \u003cp\u003e0.044\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 165px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e47.20%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e0.00%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"590\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 590px;\"\u003e\n \u003cp\u003eTable 2. Relationship between ASPSCR1 expression and tumor characteristics in patients with OS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 158px;\"\u003e\n \u003cp\u003eFeatures\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 207px;\"\u003e\n \u003cp\u003eNo. of patients\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 115px;\"\u003e\n \u003cp\u003eASPSCR1 expression\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003eAll patients\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 207px;\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 207px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003e0.034\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003e\u0026le;30years\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 207px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003e\u0026gt;30years\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 207px;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 207px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 207px;\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 207px;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eTable 3. Relationship between ASPSCR1 expression and tumor characteristics in patients with OS\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"554\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 163px;\"\u003e\n \u003cp\u003eTumor characteristics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 200px;\"\u003e\n \u003cp\u003eIndex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eASPSCR1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 163px;\"\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 200px;\"\u003e\n \u003cp\u003eSpearman correlation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003e0.334\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 163px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 200px;\"\u003e\n \u003cp\u003eSignificance (two tailed)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 163px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 200px;\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 163px;\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 200px;\"\u003e\n \u003cp\u003eSpearman correlation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003e-0.252\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 163px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 200px;\"\u003e\n \u003cp\u003eSignificance (two tailed)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"medical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"medo","sideBox":"Learn more about [Medical Oncology](https://www.springer.com/journal/12032)","snPcode":"12032","submissionUrl":"https://submission.nature.com/new-submission/12032/3","title":"Medical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8446485/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8446485/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOsteosarcoma (OS), the most common primary bone malignancy, is highly metastatic and difficult to treat. Although ASPSCR1 is implicated in multiple cancers, its role and mechanism in OS remain unclear. This study aims to clarify ASPSCR1 function in OS progression.ASPSCR1 expression and prognosis were evaluated via immunohistochemistry and statistics in clinical cohorts. Lentiviral shRNA-mediated ASPSCR1 knockdown in OS cells enabled functional assessments of proliferation, apoptosis, migration/invasion. Gene Set Enrichment Analysis (GSEA) identified pathways enrichment, further corroborated by glucose, lactate, and ATP assays. In vivo tumorigenicity was assessed using subcutaneous xenograft.ASPSCR1 was overexpressed in OS tissues and associated with poor survival. ASPSCR1 knockdown inhibited proliferation and migration while inducing apoptosis in OS cells. GSEA revealed significant enrichment of ASPSCR1-associated genes in the glycan biosynthesis. PKM2 inhibition (PKM2-IN-3) abolished ASPSCR1-driven glycolytic activity and proliferation. Consistent with in vitro findings, \u003cem\u003ein vivo\u003c/em\u003e experiments robustly supported the pivotal role of ASPSCR1 knockdown in reducing glycolytic activity and inhibiting OS tumor growth. ASPSCR1 promotes OS progression via upregulating glycolysis, representing a potential therapeutic target.\u003c/p\u003e","manuscriptTitle":"ASPSCR1 promotes osteosarcoma progression through the upregulation of glycolysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-11 19:52:16","doi":"10.21203/rs.3.rs-8446485/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-03-13T11:43:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"64270728334109672109888290200261079413","date":"2026-03-11T12:42:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-06T04:11:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-26T06:26:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-26T06:24:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Medical Oncology","date":"2025-12-25T05:23:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"medical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"medo","sideBox":"Learn more about [Medical Oncology](https://www.springer.com/journal/12032)","snPcode":"12032","submissionUrl":"https://submission.nature.com/new-submission/12032/3","title":"Medical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1bf70651-b785-4eba-9d1b-74baeafab9a9","owner":[],"postedDate":"March 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-11T19:52:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-11 19:52:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8446485","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8446485","identity":"rs-8446485","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}