PRRX1 regulates malignancies partially via regulating ANTXR1 and thus activating PI3K/mTOR signaling in hepatocarcinoma

PRRX1 regulates malignancies partially via regulating ANTXR1 and thus activating PI3K/mTOR signaling in hepatocarcinoma | 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 PRRX1 regulates malignancies partially via regulating ANTXR1 and thus activating PI3K/mTOR signaling in hepatocarcinoma Fang Wang, Qian Liu, Hongqing Chen, Tian Wen, YeKe Wu, Qiongying Hu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7814012/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Proteomic analysis of hepatocellular carcinoma (HCC) and paired adjacent non-tumor tissues revealed a significant increase in ANTXR1 phosphorylation in HCC samples. Using the GEPIA online tool, we identified a strong positive correlation between ANTXR1 and PRRX1 in HCC tissues. Moreover, the transcriptional level of PRRX1 was closely associated with overall survival in HCC patients. PRRX1, a homeodomain transcription factor, exists in two subtypes: PRRX1A and PRRX1B. These subtypes have been implicated in several malignancies, including pancreatic, breast, and lung cancers; however, their roles in HCC remain unclear. Immunohistochemical (IHC) analysis of 175 paired clinical samples demonstrated significant PRRX1 overexpression in HCC tissues compared to adjacent tissues. IIndividual overexpression of PRRX1 isoforms demonstrated that PRRX1A, but not PRRX1B, markedly promoted hepatocellular carcinoma cell proliferation. Furthermore, knockdown of PRRX1 resulted in significant downregulation of ANTXR1 protein levels in both Huh-7 and SK-HEP-1 cell lines, suggesting that PRRX1 positively regulates ANTXR1. Notably, only PRRX1A overexpression rescued the suppressed ANTXR1 transcription and altered its subcellular localization following PRRX1 knockdown. Mechanistic investigations revealed that both PRRX1 and ANTXR1 contribute to tumor progression through activation of the PI3K/Akt/mTOR signaling pathway. In summary, our results indicate that PRRX1, particularly the PRRX1A subtype, acts as a critical promoter in hepatocellular carcinoma progression. PRRX1 isoform ANTXR1 hepatocarcinoma mTOR/PI3K signaling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Paired-related homeobox 1 (PRRX1) is a transcription factor belonging to the homeodomain family [ 1 ]. It is derived from the mesoderm and plays a crucial role in a wide range of physiological and pathological processes [ 2 ]. PRRX1 is primarily involved in regulating morphogenesis and determining cell fate. It is responsible for controlling the differentiation of neural stem cells, facilitating the formation of functional pulmonary vascular networks by promoting the differentiation of endothelial cells, and contributing to epithelial-mesenchymal transition (EMT) during organogenesis in the embryo [ 3 ]. In terms of its pathological role, PRRX1 is widely recognized as a promoter of EMT and stemness in different types of solid tumors. The expression of PRRX1 is deregulated in various tumor types. It is upregulated in gastric cancer [ 4 ], colorectal cancer [ 5 ], pancreatic cancer [ 6 ], and glioblastoma [ 7 ], while downregulated in breast cancer [ 8 ], hepatocarcinoma [ 9 ], and thyroid cancer [ 10 ]. Consistent with its expression pattern, PRRX1 exhibits dual roles in tumor initiation and progression. For instance, in pancreatic cancer, PRRX1 acts as a positive regulator of stemness and EMT, contributing to poorer response to chemotherapy and prognosis. However, in breast cancer, PRRX1 has the opposite effect. Previous studies on gliomas have demonstrated that PRRX1 promotes the tumorigenicity of glioma stem cells (GSCs) and enhances the invasive properties of glioblastoma cells in laboratory settings. However, the precise role of PRRX1 in the role of PRRX1 in hepatocellular carcinoma (HCC) progression remains incompletely understood. Hepatocellular carcinoma (HCC) is a prevalent and highly lethal malignancy worldwide. Despite advances in HCC treatments, the overall survival rate of patients, particularly those in advanced stages, remains unsatisfactory due to recurrence and metastasis [ 11 ]. Radiation therapy has emerged as a viable option for locally advanced HCC, with promising results seen in new strategies like stereotactic body radiotherapy. Ionizing radiation (IR) exerts its effects by directly damaging DNA or indirectly generating free radicals through water ionization [ 12 ]. Consequently, various forms of DNA damage can occur, including single-strand breaks repaired through the base excision repair pathway and double-strand breaks (DSB) repaired through the homologous recombination pathway [ 13 ]. Unfortunately, tumor resistance to IR remains a significant impediment to effective HCC treatment. Therefore, there is an urgent need to develop novel strategies that can enhance the efficacy of radiotherapy for HCC. TEM8/ANTXR1 is a transmembrane glycoprotein with integrin-like properties that exhibits increased expression in various cancer types, tumor-associated stromal cells, and tumor-associated blood vessels [ 14 ]. Its upregulation is particularly notable in hypoxic conditions [ 15 ]. Notably, TEM8/ANTXR1 is uniquely associated with tumor vessels rather than normal blood vessels [ 16 ]. This protein has been identified as a marker for pathological, tumor-associated angiogenesis, playing a role in promoting tumor growth and potentially conferring resistance to therapies targeting angiogenesis. Recent studies have provided evidence for the enrichment of TEM8/ANTXR1 in various cancer types, including triple-negative breast cancer [ 17 ], prostate cancer [ 18 ], gastric cancer [ 19 ], pancreatic cancer [ 20 ], angiosarcoma [ 21 ], colon cancer [ 22 ], and non-small cell lung cancer (NSCLC) [ 23 ]. In multiple tumor types, the upregulation of TEM8/ANTXR1 is associated with a negative prognosis [ 20 , 22 ]. Specifically, in triple-negative breast cancer, TEM8/ANTXR1 serves as a marker of vasculogenic mimicry, which is associated with poor outcomes [ 24 ]. Vasculogenic mimicry refers to the process where tumor cells organize themselves into structures resembling endothelial cells, forming functional tubes capable of carrying red blood cells. This process is driven by hypoxia and is linked to a poor prognosis in multiple cancer types [ 25 ]. In this study, we demonstrated the expression level of PRRX1 and evaluated the effects of different isoforms of PRRX1 in progression of hepatocarcinoma. We observed the regulatory role of PRRX1 on ANTXR1 via activating PI3K/AKT/ mTORsignaling. Collectively, our study has provided a novel mechanism for hepatocarcinoma progression, suggesting that PRRX1A, one of the PRRX1 isoforms, might be a feasible predictive biomarker, which could be considered as a potential therapeutic target for hepatocarcinoma patients. 2. Material and methods 2.1 Protein profiling analysis Download the data of the PXD047213 project (18 HCC tumors and 18 adjacent normal tissues) from the PRIDE database ( https://www.ebi.ac.uk/pride/archive?sort Direction = DESC&page = 2&pageSize = 20). Use limma to analyze the differences in protein phosphorylation levels between cancer tissues and adjacent normal tissues, and visualize the results using ggplot2. 2.2 GEPIA GEPIA ( http://gepia.cancer-pku.edu.cn/ ), a public online database based on TCGA and GTEx projects, was utilized to analyze the mRNA expression levels of ANTXR1/PRRX1 in Liver Cancer and corresponding normal tissues. The differential expression was assessed using a one-way ANOVA method. The overall survival analysis was performed using the Kaplan-Meier method, and the log-rank test was applied to determine statistical significance. A p - value < 0.05 was considered statistically significant. 2.3 Immunohistochemistry From January to August 2025, tissue samples (175 pairs of cancer tissues and adjacent tissues from hepatocarcinoma patients) were collected from the biological sample bank of the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine. All experimental operations in this study were conducted in accordance with the ethical principles stipulated in the Helsinki Declaration (1975). The study protocol has been approved by the Medical Ethics Committee of Chengdu University of Traditional Chinese Medicine (Approval No: 2025016). Due to the retrospective nature of this study and the use of anonymous samples, informed consent was not required. Immunohistochemistry was performed to detect the expression of PRRX1 in liver tumor tissues. The liver tumor tissue sections were deparaffinized using xylene, dehydrated with graded concentrations of alcohol, and subjected to antigen retrieval by incubating in a retrieval solution in a microwave at high power for 30 minutes. After cooling, the slides were washed with PBS three times and sequentially incubated with rabbit anti-PRRX1 (Cat. No.: ZRB2165; Sigma-Aldrich) at a dilution of 1:50 and biotin-labeled secondary antibody (ab47844, Abcam, Cambridge, England) at a dilution of 1:1000. Finally, the slides were stained with DAB, counterstained with hematoxylin, dehydrated, cleared in xylene, and mounted. Images were captured under 200× magnification using a fluorescence microscope (X71 (U-RFL-T), Olympus, Melville, NY). 2.4 Cell cultures The human hepatocarcinoma cell lines SNU449 (FH0816, FuHeng, China), SK-HEP-1 (CL-0212, Procellsystem, China), Hep3B (SNL-082, Sunncell, China), Huh-7 (CL-0120, Procellsystem), PLC-PRF-5 (SNL-086, Sunncell) and RBE (CL-0191, Procellsystem) were stored in our laboratory. Cells were cultured in DMEM (Life Technology) supplemented with 10% FBS, 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technology). The cells were maintained at 37°C in a 5% CO 2 incubator and passaged every 3 to 4 days. 2.5 Western blot After treatment, cells were lysed via a tissue homogenizer (SC-G1, Docsense, China).The lysates were centrifuged at 12,000 g for 10 minutes at 4°C, and the supernatant was collected. The protein concentrations were measured using the Bicinchoninic Acid Kit for Protein Determination (Sigma–Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. For each sample, 20 µg of total protein was separated on a 12.5% SDS-PAGE gel and transferred onto a nitrocellulose membrane (Millipore, Billerica, MA, USA). The membrane was then incubated with the following primary antibodies: rabbit anti-PRRX1 (Cat. No.:ZRB2165; Sigma-Aldrich), Recombinant anti-DDDDK tag (Binds to FLAG® tag sequence) antibody (Cat. No.: ab205606; Abcam), rabbit anti-PI3K antibody (Cat. No.: ab2958), rabbit anti-phosphorylated PI3K TYR458 antibody (Cat. No.: ab278545), rabbit anti-mTOR antibody (Cat. No.: ab134903), rabbit anti-phosphorylated mTOR at Ser2448 (Cat. No.: ab109268), rabbit anti-β-actin (Cat. No.: ab8227; Abcam, Cambridge, England), Rabbit anti-AKT antibody (Cat. No.: ab8805, Abcam) and Rabbit anti-phosphorylated AKT at Ser473 (Cat. No.: ab285140), at a dilution of 1:1000, and incubated overnight at 4°C. The membrane was then incubated with HRP-conjugated secondary antibody (Goat anti-rabbit IgG H&L antibody, Cat.No.: ab7090, Abcam, Cambridge, England) at a dilution of 1:5000 for 2 hours at room temperature. Immunodetection was performed using ECL reagents (Cat. No.: PRN2232; GE Healthcare Bio-Sciences). 2.6 Plasmid construction For PRRX1 knockdown, the PRRX1 shRNA Plasmid (h) was purchased from Sigma-Aldrich: shScrambled (SHC016), shPRRX1 (mixture of TRCN0000020648 and TRCN0000020646).Overexpression plasmids (PRRX1A/PRRX1B/ANTXR1) were provided by Chengdu Rongyou Biotechnology Co., Ltd. 2.7 Cell viability assay Cells were suspended and adjusted to a concentration of 1×10 6 cells/ml. 5000 cells/well were plated into a 96-well plate and incubated overnight. After 24 hours, the Cell Counting Kit-8 (CCK-8, Sigma–Aldrich, St. Louis, MO, USA) solution was added and incubated for 4 hours at 37°C. The absorbance at 450 nm was measured using a microplate reader (Synergy 2 Multi-Mode Microplate Reader; BioTek, Winooski, VT, USA) to determine cell viability. 2.8 CFSE assay Cells were harvested and resuspended in serum-free PBS at a density of 1 × 10^6 cells/mL. CFSE (Thermo Fisher Scientific, C34554) was added to the cell suspension at a final working concentration of 5 µM and incubated for 20 min at 37°C in the dark. The reaction was quenched by adding 5 volumes of complete culture medium and incubating for 5 min. The cells were then washed three times with PBS and seeded into culture plates. After cultivation for the indicated periods (1, 2, 3, 4, 5 Day). Observe the cell proliferation status through a fluorescence microscope. 2.9 Plate Colony Formation Assay Cells were trypsinized into a single-cell suspension and seeded into 6-well plates at a low density (500–1000 cells per well). The cells were cultured in complete medium for 10–14 days, with the medium changed every 3–4 days. After formation of macroscopic colonies, the cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min, and then stained with 0.1% crystal violet (Beyotime, C0121) for 15 min at room temperature. The plates were rinsed with tap water and air-dried. Colonies containing more than 50 cells were photographed and counted manually. 2.10 Soft Agar Colony Formation Assay A base layer of 0.6% low-melting-point agarose (Invitrogen, 16520050) in complete medium was prepared in 6-well plates and allowed to solidify. A top layer of 0.3% agarose containing 5 × 10^3 cells was plated on top of the base layer. After the top layer solidified, 1 mL of complete medium was added on top and replaced twice a week. The plates were incubated for 3–4 weeks at 37°C. Colonies were stained with 0.005% Crystal Violet for 1 h and then imaged under a microscope. The number of colonies was quantified using ImageJ software. 2.11 Cell invasion assay using Transwell Matrigel (Millipore Corporation, Darmstadt, Germany) was diluted 1:2 in DMEM/F-12, and 60 µL of the diluted Matrigel was added to the upper chamber of the Transwell plates. The chambers were then incubated at 37°C in a 5% CO2 incubator for 2 hours. Single cells were resuspended in DMEM/F-12 and seeded into the upper chamber at a concentration of 2×104 cells/well. Subsequently, 600 µL of DMEM was added to the lower chamber. After 24 hours of incubation, the inserts were collected, and the cells on the lower surface were fixed in 4% paraformaldehyde and stained with crystal violet for 15 minutes. The cells in five random fields were counted. 2.12 Immunofluorescence Staining Cells grown on glass coverslips were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 5% BSA for 1 h at room temperature. The cells were then incubated with primary antibody (anti-ANTXR1, 1:400, Abcam, ab241067) overnight at 4°C. After washing with PBS, the cells were incubated with a fluorophore-conjugated secondary antibody (FITC, 1:500, Invitrogen, F-2765) and DAPI (1 µg/mL, Beyotime, C1002) for 1 h at room temperature in the dark. The coverslips were mounted onto glass slides with antifade mounting medium (Beyotime, P0126). Images were captured using a confocal laser scanning microscope (Zeiss LSM 880). 2.13 RT-qPCR Total RNA was extracted from cells using TRIzol reagent (Invitrogen, 15596026) according to the manufacturer's protocol. cDNA was synthesized from 1 µg of total RNA using a HiScript II Q RT SuperMix kit (Vazyme, R223-01). Quantitative PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02) on a QuantStudio 5 Real-Time PCR System (Applied Biosystems). The relative mRNA expression levels were calculated using the 2^(-ΔΔCt) method and normalized to the expression of GAPDH as an internal control. The primer sequences used were as follows:PRRX1 F-5’-TGATGCTTTTGTGCGAGAA GA-3’;R-5’-AGGGAAGC GTTTTTATTGGCT-3’.ANTXR1 F-5′-ACAGTTGGCTCACAAATTCATCA-3′༛R-5′-TCACTG GCCCTTTCAAATCCT-3′.GAPDH F-5’-GCACCGTCAAGGCTGA GAAC-3’༛R-5’-TGGTGAAGACGCCAGTGGA-3’. 2.14 Flow Cytometry for Cell Cycle Analysis Cells were harvested, washed with PBS, and fixed in 70% ice-cold ethanol overnight at -20°C. The fixed cells were washed with PBS and then incubated with RNase A (100 µg/mL, Beyotime, ST577) and propidium iodide (PI, 50 µg/mL, Beyotime, ST511) for 30 min at 37°C in the dark. The DNA content of the stained cells was analyzed by flow cytometry (BD FACS Celesta). The cell cycle distribution (G0/G1, S, and G2/M phases) was determined using the ModFit LT software (v5.0). 2.15 Statistical analysis Statistical analysis was performed using SPSS version 13.0. The data are expressed as mean ± standard deviation (SD; x ± s). The statistical significance of differences between groups was assessed using Student’s t-tests and one-way analysis of variance followed by Bonferroni post hoc analysis. A p-value < 0.05 was considered statistically significant. 3. Results 3.1 In hepatocellular carcinoma, the expression level of PRRX1 is elevated. Protein proteomic analysis of liver cancer tissues and adjacent tissues revealed that the phosphorylation level of ANTXR1 in cancer tissues was significantly higher than that in adjacent tissues (Fig. 1 ). However, ANTXR1 expression showed no significant correlation with overall survival in HCC patients (Fig. 2 -A). Through analysis using the GEPIA online tool, we discovered that the transcriptional level of ANTXR1 was closely associated with PRRX1, and this association was notably stronger in cancer tissues compared to normal tissues (Fig. 2 -B). Additionally, PRRX1 was found to significantly shorten the overall survival of liver cancer patients (Fig. 2 -C). PRRX1 has been shown to play a role in promoting malignant behaviors in multiple types of tumors, such as lung cancer. To explore the impact of PRRX1 on the development of liver cancer, we initially conducted IHC assays for PRRX1 on 175 pairs of hepatocellular carcinoma pathological specimens (Fig. 2 -D). The results indicated that PRRX1 was predominantly expressed in the cytoplasm, and the protein level of PRRX1 was significantly elevated in cancer tissues. 3.2 The isoforms of PRRX1 promote the proliferation and malignant behavior of hepatocellular carcinoma cells. To explore the function of PRRX1 in hepatocellular carcinoma cell lines, we employed RT-qPCR and Western Blot techniques to detect the mRNA and protein levels of PRRX1 in SNU449, SK-HEP-1, Hep3B, Huh-7, PLC-PRF-5, and RBE cell lines. The results indicated that although there were no substantial differences in the mRNA levels of PRRX1 among these cell lines, the protein levels of PRRX1 were remarkably higher in Huh-7 and SK-HEP-1 cells (Fig. 3 -A, B). Subsequently, Huh- 7 and SK-HEP-1 cell lines were selected for subsequent experiments to investigate the tumor-regulatory functions of PRRX1. PRRX1 is a protein consisting of two subtypes, namely PRRX1A and PRRX1B. Previous reports have demonstrated that PRRX1A is involved in promoting the malignant phenotypes of lung cancer. To determine whether the function of PRRX1 in hepatocellular carcinoma is subtype-dependent, we respectively overexpressed FLAG-tagged PRRX1A and FLAG-tagged PRRX1B in Huh-7 and SK-HEP-1 cells (Fig. 3 -C). The results of the CCK-8 assay showed that while the proliferation rates of cells transfected with either the vector or FLAG-PRRX1B did not exhibit significant alterations, the overexpression of FLAG-PRRX1A significantly accelerated cell proliferation (Fig. 3 -D). To further verify this, CFSE live-cell staining was performed, and cell proliferation was monitored under a fluorescence microscope. The results further confirmed that the overexpression of FLAG-PRRX1A, rather than FLAG-PRRX1B, promoted the proliferation of Huh-7 and SK-HEP-1 cells (Fig. 3 -E, F). To further elucidate the impact of PRRX1A overexpression on tumor malignancy, we examined the effects of FLAG-PRRX1A overexpression on the migration and invasion capabilities of tumor cells (Fig. 4 -A), tumorigenesis (Fig. 4 -B), and colony - forming ability (Fig. 4 -C). As anticipated, the overexpression of FLAG-PRRX1A significantly enhanced the malignant behaviors of tumor cells. 3.3 PRRX1 transcriptionally upregulates ANTXR1. To precisely define the interplay between PRRX1 and ANTXR1, we carried out Western blot analyses. The findings revealed that upon knockdown of PRRX1, the protein level of ANTXR1 was downregulated. Conversely, knockdown of ANTXR1 did not impact the protein level of PRRX1. Additionally, simultaneous overexpression of ANTXR1 during PRRX1 knockdown was able to reverse the decline in ANTXR1 protein levels induced by PRRX1 knockdown (Fig. 5 -A). To investigate whether PRRX1 influences the membrane localization of ANTXR1, we performed immunofluorescence experiments. The results demonstrated that shRNA-mediated knockdown of PRRX1 significantly decreased the membrane-localized ANTXR1 protein levels in both Huh-7 and SK-HEP-1 cells. This finding indicates that PRRX1 is essential for maintaining the stability of ANTXR1. It is noteworthy that in cells lacking PRRX1, ectopic overexpression of ANTXR1 fully restored its membrane localization. This suggests that PRRX1 regulates ANTXR1 at the post-transcriptional level, rather than being solely associated with its expression (Fig. 5 -B). This functional interaction implies that PRRX1 may modulate ANTXR1 through mechanisms such as protein stabilization, co-transport, or transcriptional activation pathways. 3.4 PRRX1A, but not PRRX1B transcriptional upregulates ANTXR1 and its membrane location. To elucidate the role of PRRX1 subtypes in modulating ANTXR1 transcription and membrane localization, we conducted RT-qPCR analyses following the overexpression of PRRX1A or PRRX1B in cells where PRRX1 had been knocked down. The results demonstrated that the overexpression of PRRX1A significantly upregulated the mRNA levels of ANTXR1, whereas PRRX1B did not exhibit such an effect (Fig. 6 -A). This finding indicates that the homologous isoform PRRX1A specifically trans-activates ANTXR1. In line with this observation, confocal microscopy analysis showed that the overexpression of PRRX1A substantially enhanced the membrane-localized ANTXR1 protein levels in cells lacking PRRX1. In contrast, the overexpression of PRRX1B was unable to rescue the membrane localization of ANTXR1 (Fig. 6 -B). Collectively, these data suggest that PRRX1A uniquely orchestrates the transcriptional activation and membrane trafficking of ANTXR1, thereby highlighting its pivotal functional role within the PRRX1-ANTXR1 axis. 3. 5 PRRX1 activates the PI3K/Akt/mTOR signaling pathway by activating ANTXR1. Cai and his colleagues reported that ANTXR1 serves as a crucial activator of the PI3K/AKT/mTOR signaling pathway, thereby facilitating cancer progression [ 26 ]. We postulated that PRRX1 might activate the PI3K/AKT/mTOR signaling pathway through the upregulation of ANTXR1. In Huh-7 and SK-HEP-1 cells, interfering with either ANTXR1 or PRRX1 led to a suppression of the phosphorylation levels of PI3K/AKT/mTOR. Nevertheless, following the interference of PRRX1, the subsequent introduction of exogenous ANTXR1 reactivated the phosphorylation levels of PI3K/AKT/mTOR. This finding indicates that PRRX1 activates the PI3K/AKT/mTOR signaling pathway via the upregulation of ANTXR1 (Fig. 7 -A).To further validate this mechanism, we added LY294002, a specific inhibitor of ANTXR1, to cells in which PRRX1 had been interfered with and then exogenous ANTXR1 had been introduced. The results demonstrated that the addition of LY294002 once again inhibited the phosphorylation levels of PI3K/AKT/mTOR (Fig. 7 -B). Cell cycle analysis revealed that interfering with PRRX1 caused cell cycle arrest at the G1/G0 phase, and this effect could be reversed by the overexpression of ANTXR1 (Fig. 7 -C). Furthermore, Western blot analysis results indicated that ANTXR1 may influence cell proliferation capacity by upregulating Cyclin D1 and P21 while downregulating P27 (Fig. 7 -D). 4. Discussion Our study demonstrates that PRRX1 is significantly upregulated in HCC tissues and transcriptionally regulates ANTXR1. Functional assays revealed that the PRRX1A isoform, but not PRRX1B, exerts tumor-promoting effects in HCC cells. Although we identified that PRRX1 promotes HCC malignancy via ANTXR1, the precise mechanistic details of this regulation warrant further investigation. PRRX1 (Paired Related Homeobox 1) is a transcription factor that plays important biological functions in embryonic development and cell differentiation. PRRX1 plays a crucial role in embryonic development, contributing to the formation of various organs and tissues such as the heart, kidneys, lungs, and skeletal system. PRRX1 promotes the normal formation and development of tissues and organs by regulating gene expression and participating in cell fate determination and cell migration. PRRX1 is involved in the regulation of cell differentiation, transforming cells from a primitive state into specific cell types. PRRX1 also has an impact on the maintenance and differentiation of embryonic stem cells, which are cells with self-renewal and differentiation potential. PRRX1’s role in tumorigenesis and tumor development is completely different. The expression and function of PRRX1 in breast cancer have received extensive attention. Studies have shown that PRRX1 may be involved in the proliferation, invasion, and metastasis of breast cancer cells. It may serve as a potential therapeutic target or diagnostic marker for breast cancer. However, the specific mechanisms and regulatory networks of PRRX1 still require further research for a comprehensive understanding. Isoforms are protein variants that arise from the same gene with slight differences. They may exhibit variations in structure, function, and expression. PRRX1 has two isoforms, PRRX1A and PRRX1B, which display significant functional differences. PRRX1A and PRRX1B are involved in regulating the processes of epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) [ 27 , 28 ], which modulate metastasis in hepatocarcinomas (29). This isoform-specific regulation of the EMT-MET axis has been revealed in breast (30), colorectal (31, 32), and gastric cancer (33). In our results, PRRX1A exerts promoting effects on proliferation, invasion, colony formation and tumor formation in soft agar, but not PRRX1B, in hepatocarcinoma cells. We also evaluated the transcriptional regulation of PRRX1 on ANTXR1. However, it is still unclear that whether the regulation is dependent on different isoforms, which is worth to be investigated in further study. ANTXR1 (Anthrax Toxin Receptor 1), also known as TEM8 (Tumor endothelial marker 8), is a transmembrane protein closely involved in the regulation of multiple signaling pathways and cellular functions. ANTXR1 interacts with key proteins in the mTOR/PI3K signaling pathway. For example, ANTXR1 can form a complex with PI3K (Phosphoinositide 3-kinase) and promote the activation of the PI3K signaling pathway. This interaction may be achieved through specific domains within the cytoplasmic region of ANTXR1. Furthermore, the activation of ANTXR1 can influence processes such as cell proliferation, survival, and metabolism through the PI3K/AKT/mTOR signaling pathway. The interaction between ANTXR1 and PI3K activates PI3K, subsequently activating downstream proteins such as AKT (Protein Kinase B) and mTOR (Mammalian Target of Rapamycin), promoting cell proliferation and growth. In the context of cancer, abnormal activation of the ANTXR1-mTOR/PI3K signaling pathway plays a crucial role in the development of various types of cancers. High expression of ANTXR1 is associated with cancer cell growth, invasion, and metastasis, potentially promoting tumor cell proliferation and survival through the PI3K/AKT/mTOR signaling pathway. 5. Conclusions In our experiments, we observed that interference with PRRX1 and ANTXR1 can inhibit the mTOR/PI3K signaling pathway. This inhibition occurs by suppressing the phosphorylation of key proteins, indicating the essential role of PRRX1/ANTXR1 in the activity of mTOR/PI3K and subsequently affecting cell proliferation and the cell cycle progression. In conclusion, we speculate that PRRX1 likely exerts as a tumor promoter in hepatocarcinoma. However, further study is required to clarify the mechanism. Declarations Author Contributions Q.Y.H. and Y.K.W. conceived and designed the study. F.W. and Q.L. performed the cellular experiments. H.Q.C. and T.W. conducted the animal experiments. Q.Y.H. and Y.K.W. wrote and revised the manuscript. All authors have read and approved the final manuscript. Ethics Approval and Consent to Participate The human tissue-related experiments in this study were approved by the Ethics Committee of Chengdu University of Traditional Chinese Medicine (Approval No. 2025016) and were conducted in strict accordance with the Declaration of Helsinki. As this study was a retrospective analysis utilizing anonymized samples, the requirement for informed consent was waived by the Chengdu University of Traditional Chinese Medicine Ethics Committee. Acknowledgment The authors would like to thank Professor Huimin Shi (Peking University, Beijing, China) for language editing. Funding This study was supported by National Natural Science Foundation of China (NSFC, 81973684), 2023NSFSC1760 and Xinglin scholar (QJRC2022007) Data access statement The data analyzed in FIG1 is obtained from the PRIDE database(PXD047213, https://www.ebi.ac.uk/pride/archive/projects/PXD047213). The data analyzed in FIG2-A-C is from the TCGA database and was generated through GEPIA (http://gepia.cancer-pku.cn/).The other data generated in this study are available upon request from the corresponding author. Conflict of Interest The authors declare no competing interests. Consent for publication All authors reviewed and approved the final manuscript. Qiongying Hu is responsible for the overall content as the guarantor. References Shimozaki K, Clemenson GD, Jr., Gage FH. Paired related homeobox protein 1 is a regulator of stemness in adult neural stem/progenitor cells. J. Neurosci. 2013;33:4066–4075. Ihida-Stansbury K, et al. Paired-related homeobox gene Prx1 is required for pulmonary vascular development. Circ. Res. 2004;94:1507–1514. Ocana OH, et al. Metastatic colonization requires the repression of the epithelial-mesenchymal transition inducer PRRX1. Cancer Cell. 2012;22:709–724. Guo J, et al. PRRX1 promotes epithelial-mesenchymal transition through the Wnt/β-catenin pathway in gastric cancer. Med. Oncol.2015;32:393. Takahashi Y, et al. Paired related homoeobox 1, a new EMT inducer, is involved in metastasis and poor prognosis in colorectal cancer. Brit J. Cancer. 2013;109:307–311. Marchand B, et al. PRRX1 isoforms cooperate with FOXM1 to regulate the DNA damage response in pancreatic cancer cells. Oncogene.2019;38:4325–4339. Li Y, et al. Paired related homeobox 1 transactivates dopamine D2 receptor to maintain propagation and tumorigenicity of glioma-initiating cells. J. Mol. Cell Biol. 2017;9:302–314. Ocana OH, et al. Metastatic colonization requires the repression of the epithelial-mesenchymal transition inducer PRRX1. Cancer Cell. 2012;22:709–724. Fan M, et al. Downregulation of PRRX1 via the p53-dependent signaling pathway predicts poor prognosis in hepatocellular carcinoma. Oncol. Rep. 2017;38:1083–1090. Hardin H, et al. The roles of the epithelial-mesenchymal transition marker PRRX1 and miR-146b-5p in papillary thyroid carcinoma progression. Am. J. Pathol. 2014;184:2342–2354. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65(2):87–108. Liang XH, Loncarevic IF, Tang ZY, Yu YQ, Zentgraf H, Schroder CH. Resection of hepatocellular carcinoma: oligocentric origin of recurrent and multinodular tumours. J Gastroenterol Hepatol. 1991;6(1):77–80. Chambers AF, Groom AC, MacDonald IC. Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer. 2002;2(8):563–572. Yang M. Y., Chaudhary A., Seaman S., Dunty J., Stevens J., Elzarrad M. K., et al. (2011). The cell surface structure of tumor endothelial marker 8 (tem8) is regulated by the actin cytoskeleton. Biochim. Biophys. Acta 1813, 39–49. Opoku-Darko M., Yuen C., Grassi C., Gratton K., Bathe O. (2007). P54. J. Surg. Res. 137, 260. Chaudhary A., Hilton M. B., Seaman S., Haines D. C., Stevenson S., Lemotte P. K., et al. (2012). Tem8/antxr1 blockade inhibits pathological angiogenesis and potentiates tumoricidal responses against multiple cancer types. Cancer Cell 21, 212–226. Xu J., Yang X., Deng Q., Yang C., Wang D., Jiang G., et al. (2021). Tem8 marks neovasculogenic tumor-initiating cells in triple-negative breast cancer. Nat. Commun. 12, 4413. Li M., Fang L., Kwantwi L. B., He G., Luo W., Yang L., et al. (2021). N-myc promotes angiogenesis and therapeutic resistance of prostate cancer by tem8. Med. Oncol. 38, 127. Sun K-R., Lv H-F., Chen B-B., Nie C. Y., Zhao J., Chen X. B. (2021). Latest therapeutic target for gastric cancer: Anthrax toxin receptor 1. World J. Gastrointest. Oncol. 13, 216–222. Alcalá S., Martinelli P., Hermann P. C., Heeschen C., Sainz B. (2019). The anthrax toxin receptor 1 (antxr1) is enriched in pancreatic cancer stem cells derived from primary tumor cultures. Stem Cells Int. 2019, 1378639. Kusaba Y., Kajihara I., Sakamoto R., Maeda-Otsuka S., Yamada-Kanazawa S., Sawamura S., et al. (2021). Overexpression of tumor endothelial marker 8 protein predicts poor prognosis in angiosarcoma. J. Dermatol. 48, E514–E516. Ł Pietrzyk, Korolczuk A., Matysek M., Arciszewski M. B., Torres K. (2021). Clinical value of detecting tumor endothelial marker 8 (antxr1) as a biomarker in the diagnosis and prognosis of colorectal cancer. Cancer Manag. Res. 13, 3113–3122. Gong Q., Deng J., Zhang L., Zhou C., Fu C., Wang X., et al. (2021). Targeted silencing of tem8 suppresses non-small cell lung cancer tumor growth via the erk/bcl-2 signaling pathway. Mol. Med. Rep. 24, 595. Fernández-Cortés M., Delgado-Bellido D., Oliver F. J. (2019). Vasculogenic mimicry: Become an endothelial cell "but not so much. Front. Oncol. 9, 803. Chen D., Bhat-Nakshatri P., Goswami C., Badve S., Nakshatri H. (2013). Antxr1, a stem cell-enriched functional biomarker, connects collagen signaling to cancer stem-like cells and metastasis in breast cancer. Cancer Res. 73, 5821–5833. Cai C, Dang W, Liu S, et al. Anthrax toxin receptor 1/tumor endothelial marker 8 promotes gastric cancer progression through activation of the PI3K/AKT/mTOR signaling pathway. Cancer Sci. 2020;111(4):1132-1145. Marchand B, Pitarresi JR, Reichert M, et al. PRRX1 isoforms cooperate with FOXM1 to regulate the DNA damage response in pancreatic cancer cells. Oncogene 2019;38:4325-39. Ocaña OH, Coskun H, Minguillón C, et al. A right-handed signalling pathway drives heart looping in vertebrates. Nature 2017;549:86-90. Takano S, Reichert M, Bakir B, et al. PRRX1 isoform switching regulates pancreatic cancer invasion and metastatic colonization. Genes Dev 2016;30:233-47. Ocaña OH, Córcoles R, Fabra A, et al. Metastatic colonization requires the repression of the epithelial-mesenchymal transition inducer PRRX1. Cancer Cell 2012;22:709-24. Takahashi Y, Sawada G, Kurashige J, et al. Paired related homoeobox 1, a new EMT inducer, is involved in metastasis and poor prognosis in colorectal cancer. Br J Cancer 2013;109:307-11. Zheng L, Zhang Y, Lin S, et al. Down-regualtion of miR-106b induces epithelial-mesenchymal transition but suppresses metastatic colonization by targeting PRRX1 in colorectal cancer. Int J Clin Exp Pathol 2015;8:10534-44. Ellsworth RE, Blackburn HL, Shriver CD, et al. Molecular heterogeneity in breast cancer: State of the science and implications for patient care. Semin Cell Dev Biol 2017;64:65-72. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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-7814012","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":554785489,"identity":"8ea93c38-6774-406a-ae07-f54fdc57c0f8","order_by":0,"name":"Fang Wang","email":"","orcid":"","institution":"Chengdu University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Fang","middleName":"","lastName":"Wang","suffix":""},{"id":554785490,"identity":"4048740d-b733-4cc9-bdf7-7dccfc72b363","order_by":1,"name":"Qian Liu","email":"","orcid":"","institution":"Chengdu University of Traditional Chinese 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Heatmap of the top 50 proteins exhibiting differences in phosphorylation levels between hepatocellular cancer tissues and adjacent tissues.\u003c/p\u003e\n\u003cp\u003eB. Volcano plot of the proteins exhibiting differences in phosphorylation levels between hepatocellular cancer tissues and adjacent tissues.\u003c/p\u003e\n\u003cp\u003eC. The protein structure of ANTXR1.\u003c/p\u003e\n\u003cp\u003eD. The phosphorylation level of ANTXR1 differed between hepatocellular cancer tissues and adjacent tissues.\u003c/p\u003e","description":"","filename":"figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7814012/v1/cf28eea89483daf74d3c066b.jpg"},{"id":97658843,"identity":"8c323772-9aae-4868-b821-69cbe0881abf","added_by":"auto","created_at":"2025-12-08 07:33:49","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1263366,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePRRX1 affects the survival period of hepatocellular carcinoma and is highly expressed in hepatocellular cancer tissues.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. The influence of the transcriptional level of ANTXR1 on the survival period of liver cancer patients.\u003c/p\u003e\n\u003cp\u003eB. Correlation analysis of the transcriptional level of ANTXR1 and PRRX1.\u003c/p\u003e\n\u003cp\u003eC. The influence of the transcriptional level of PRRX1 on the survival period of liver cancer patients.\u003c/p\u003e\n\u003cp\u003eD. 175 cases of IHC analysis of PRRX1 in hepatocellular cancer tissues and adjacent tissues of patients.\u003c/p\u003e","description":"","filename":"figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7814012/v1/cfe389fafd9d0d44a2e626ee.jpg"},{"id":97674273,"identity":"3cfae1a0-158b-45b1-8318-744449eef411","added_by":"auto","created_at":"2025-12-08 09:42:51","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1651196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effects of isoforms of PRRX1A and PRRX1B in Huh-7 and SK-HEP-1 cells on cell viability.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. The mRNA levels of PRRX1 in different hepatocellular cancer cell lines.\u003c/p\u003e\n\u003cp\u003eB. The protein levels of PRRX1 in different hepatocellular cancer cell lines.\u003c/p\u003e\n\u003cp\u003eC. After stably transfection of Flag-PRRX1A or Flag-PRRX1B in Huh-7 and SK-HEP-1 cells, Flag-PRRX1A or Flag-PRRX1B was detected by western blot.\u003c/p\u003e\n\u003cp\u003eD. After stably transfection of Flag-PRRX1A or Flag-PRRX1B in Huh-7 and SK-HEP-1 cells, cell viability was detected by CCK-8 assay.\u003c/p\u003e\n\u003cp\u003eE. Using CFSE for live cell staining, and observing the cell proliferation of Huh-7 cells overexpressing PRRX1A or PRRX1B under a fluorescence microscope.\u003c/p\u003e\n\u003cp\u003eF. Using CFSE for live cell staining, and observing the cell proliferation of SK-HEP-1 cells overexpressing PRRX1A or PRRX1B under a fluorescence microscope.\u003c/p\u003e","description":"","filename":"figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7814012/v1/80042cb213493d113cce029e.jpg"},{"id":97674212,"identity":"725e3273-bbab-46e9-bcc1-05711c68a534","added_by":"auto","created_at":"2025-12-08 09:42:38","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2437365,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effects of isoforms of PRRX1A and PRRX1B in Huh-7 and SK-HEP-1 cells on invasion, colony formation and tumor formation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter stably transfection of Flag-PRRX1A or Flag-PRRX1B in Huh-7 and SK-HEP-1 cells, invasion (A), tumor formation in soft agar (B) and colony formation (C) were detected.\u003c/p\u003e","description":"","filename":"figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7814012/v1/ee0f8d133b889b4c868a330b.jpg"},{"id":97674715,"identity":"bd8eb3e5-521d-49dc-adf5-926159f35f5b","added_by":"auto","created_at":"2025-12-08 09:43:57","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3750478,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe interrelationship between PRRX1 and ANTXR1 in Huh-7 and SK-HEP-1 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. After stably knockdown of PRRX1 or ANTXR1, protein levels of PRRX1 or ANTXR1 were measured by performing western blot.\u003c/p\u003e\n\u003cp\u003eB. Knockdown of ANTXR1 (shANTXR1) or PRRX1 (shPRRX1) in Huh-7 and SK-HEP-1 cells significantly reduced membrane-localized ANTXR1 protein levels, as demonstrated by confocal microscopy.\u003c/p\u003e","description":"","filename":"figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7814012/v1/4034ce5433b9b3393bed1270.jpg"},{"id":97675036,"identity":"f27e32b2-b581-4842-bf80-ee1ef9570e03","added_by":"auto","created_at":"2025-12-08 09:45:40","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3810504,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePRRX1A, but not PRRX1B, regulates ANTXR1 transcriptionally.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. RT-qPCR analysis of ANTXR1 mRNA levels in Huh-7 and SK-HEP-1 cells after PRRX1 knockdown and rescue with PRRX1A or PRRX1B.\u003c/p\u003e\n\u003cp\u003eB. Confocal microscopy of ANTXR1 membrane localization in PRRX1-depleted cells rescued with PRRX1A or PRRX1B. Scale bar = 20 μm. ​​\u003c/p\u003e","description":"","filename":"figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7814012/v1/0fa8dfb153100b47b1c58b6e.jpg"},{"id":97658852,"identity":"93dd7e0c-34d4-4fbb-be1d-99625cef30a7","added_by":"auto","created_at":"2025-12-08 07:33:49","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":894848,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eknockdown of PRRX1 inactivates PI3K/AKT/mTOR signaling potentially via ANTXR1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. After knockdown of ANTXR1 or PRRX1, hallmarkers of PI3K/AKT/mTOR signaling were detected by performing western blot.\u003c/p\u003e\n\u003cp\u003eB. After overexpression of ANTXR1 with or without addition of LY294002, hallmarkers of PI3K/AKT/mTOR signaling were detected by performing Western Blot.\u003c/p\u003e\n\u003cp\u003eC. After PRRX1 or ANTXR1 knockdown, the cell cycle phases distribution in Huh-7 or SK-HEP-1 cells.\u003c/p\u003e\n\u003cp\u003eD. After PRRX1 or ANTXR1 knockdown, the cell proliferation-related protein, including Cyclin D1, p27 and p21 were measured.\u003c/p\u003e","description":"","filename":"figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7814012/v1/a3292eeab5be46c836e97c33.jpg"},{"id":102415541,"identity":"8b4b506d-d77c-43f9-9e42-879a05f7338d","added_by":"auto","created_at":"2026-02-11 12:44:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16111004,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7814012/v1/8c41739b-fa04-44e9-9197-a4e735e8bcdf.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"PRRX1 regulates malignancies partially via regulating ANTXR1 and thus activating PI3K/mTOR signaling in hepatocarcinoma","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePaired-related homeobox 1 (PRRX1) is a transcription factor belonging to the homeodomain family [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is derived from the mesoderm and plays a crucial role in a wide range of physiological and pathological processes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. PRRX1 is primarily involved in regulating morphogenesis and determining cell fate. It is responsible for controlling the differentiation of neural stem cells, facilitating the formation of functional pulmonary vascular networks by promoting the differentiation of endothelial cells, and contributing to epithelial-mesenchymal transition (EMT) during organogenesis in the embryo [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In terms of its pathological role, PRRX1 is widely recognized as a promoter of EMT and stemness in different types of solid tumors.\u003c/p\u003e\u003cp\u003eThe expression of PRRX1 is deregulated in various tumor types. It is upregulated in gastric cancer [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], colorectal cancer [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], pancreatic cancer [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and glioblastoma [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], while downregulated in breast cancer [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], hepatocarcinoma [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and thyroid cancer [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Consistent with its expression pattern, PRRX1 exhibits dual roles in tumor initiation and progression. For instance, in pancreatic cancer, PRRX1 acts as a positive regulator of stemness and EMT, contributing to poorer response to chemotherapy and prognosis. However, in breast cancer, PRRX1 has the opposite effect. Previous studies on gliomas have demonstrated that PRRX1 promotes the tumorigenicity of glioma stem cells (GSCs) and enhances the invasive properties of glioblastoma cells in laboratory settings. However, the precise role of PRRX1 in the role of PRRX1 in hepatocellular carcinoma (HCC) progression remains incompletely understood.\u003c/p\u003e\u003cp\u003eHepatocellular carcinoma (HCC) is a prevalent and highly lethal malignancy worldwide. Despite advances in HCC treatments, the overall survival rate of patients, particularly those in advanced stages, remains unsatisfactory due to recurrence and metastasis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Radiation therapy has emerged as a viable option for locally advanced HCC, with promising results seen in new strategies like stereotactic body radiotherapy. Ionizing radiation (IR) exerts its effects by directly damaging DNA or indirectly generating free radicals through water ionization [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Consequently, various forms of DNA damage can occur, including single-strand breaks repaired through the base excision repair pathway and double-strand breaks (DSB) repaired through the homologous recombination pathway [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Unfortunately, tumor resistance to IR remains a significant impediment to effective HCC treatment. Therefore, there is an urgent need to develop novel strategies that can enhance the efficacy of radiotherapy for HCC.\u003c/p\u003e\u003cp\u003eTEM8/ANTXR1 is a transmembrane glycoprotein with integrin-like properties that exhibits increased expression in various cancer types, tumor-associated stromal cells, and tumor-associated blood vessels [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Its upregulation is particularly notable in hypoxic conditions [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Notably, TEM8/ANTXR1 is uniquely associated with tumor vessels rather than normal blood vessels [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This protein has been identified as a marker for pathological, tumor-associated angiogenesis, playing a role in promoting tumor growth and potentially conferring resistance to therapies targeting angiogenesis.\u003c/p\u003e\u003cp\u003eRecent studies have provided evidence for the enrichment of TEM8/ANTXR1 in various cancer types, including triple-negative breast cancer [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], prostate cancer [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], gastric cancer [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], pancreatic cancer [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], angiosarcoma [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], colon cancer [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and non-small cell lung cancer (NSCLC) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In multiple tumor types, the upregulation of TEM8/ANTXR1 is associated with a negative prognosis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Specifically, in triple-negative breast cancer, TEM8/ANTXR1 serves as a marker of vasculogenic mimicry, which is associated with poor outcomes [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Vasculogenic mimicry refers to the process where tumor cells organize themselves into structures resembling endothelial cells, forming functional tubes capable of carrying red blood cells. This process is driven by hypoxia and is linked to a poor prognosis in multiple cancer types [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, we demonstrated the expression level of PRRX1 and evaluated the effects of different isoforms of PRRX1 in progression of hepatocarcinoma. We observed the regulatory role of PRRX1 on ANTXR1 via activating PI3K/AKT/ mTORsignaling. Collectively, our study has provided a novel mechanism for hepatocarcinoma progression, suggesting that PRRX1A, one of the PRRX1 isoforms, might be a feasible predictive biomarker, which could be considered as a potential therapeutic target for hepatocarcinoma patients.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Protein profiling analysis\u003c/h2\u003e\u003cp\u003eDownload the data of the PXD047213 project (18 HCC tumors and 18 adjacent normal tissues) from the PRIDE database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/pride/archive?sort\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/pride/archive?sort\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e Direction\u0026thinsp;=\u0026thinsp;DESC\u0026amp;page\u0026thinsp;=\u0026thinsp;2\u0026amp;pageSize\u0026thinsp;=\u0026thinsp;20). Use limma to analyze the differences in protein phosphorylation levels between cancer tissues and adjacent normal tissues, and visualize the results using ggplot2.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 GEPIA\u003c/h2\u003e\u003cp\u003eGEPIA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.edu.cn/\u003c/span\u003e\u003cspan address=\"http://gepia.cancer-pku.edu.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), a public online database based on TCGA and GTEx projects, was utilized to analyze the mRNA expression levels of ANTXR1/PRRX1 in Liver Cancer and corresponding normal tissues. The differential expression was assessed using a one-way ANOVA method. The overall survival analysis was performed using the Kaplan-Meier method, and the log-rank test was applied to determine statistical significance. A p - value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Immunohistochemistry\u003c/h2\u003e\u003cp\u003eFrom January to August 2025, tissue samples (175 pairs of cancer tissues and adjacent tissues from hepatocarcinoma patients) were collected from the biological sample bank of the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine. All experimental operations in this study were conducted in accordance with the ethical principles stipulated in the Helsinki Declaration (1975). The study protocol has been approved by the Medical Ethics Committee of Chengdu University of Traditional Chinese Medicine (Approval No: 2025016). Due to the retrospective nature of this study and the use of anonymous samples, informed consent was not required.\u003c/p\u003e\u003cp\u003eImmunohistochemistry was performed to detect the expression of PRRX1 in liver tumor tissues. The liver tumor tissue sections were deparaffinized using xylene, dehydrated with graded concentrations of alcohol, and subjected to antigen retrieval by incubating in a retrieval solution in a microwave at high power for 30 minutes. After cooling, the slides were washed with PBS three times and sequentially incubated with rabbit anti-PRRX1 (Cat. No.: ZRB2165; Sigma-Aldrich) at a dilution of 1:50 and biotin-labeled secondary antibody (ab47844, Abcam, Cambridge, England) at a dilution of 1:1000. Finally, the slides were stained with DAB, counterstained with hematoxylin, dehydrated, cleared in xylene, and mounted. Images were captured under 200\u0026times; magnification using a fluorescence microscope (X71 (U-RFL-T), Olympus, Melville, NY).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Cell cultures\u003c/h2\u003e\u003cp\u003eThe human hepatocarcinoma cell lines SNU449 (FH0816, FuHeng, China), SK-HEP-1 (CL-0212, Procellsystem, China), Hep3B (SNL-082, Sunncell, China), Huh-7 (CL-0120, Procellsystem), PLC-PRF-5 (SNL-086, Sunncell) and RBE (CL-0191, Procellsystem) were stored in our laboratory. Cells were cultured in DMEM (Life Technology) supplemented with 10% FBS, 100 units/ml penicillin, and 100 \u0026micro;g/ml streptomycin (Life Technology). The cells were maintained at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator and passaged every 3 to 4 days.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Western blot\u003c/h2\u003e\u003cp\u003eAfter treatment, cells were lysed via a tissue homogenizer (SC-G1, Docsense, China).The lysates were centrifuged at 12,000 g for 10 minutes at 4\u0026deg;C, and the supernatant was collected. The protein concentrations were measured using the Bicinchoninic Acid Kit for Protein Determination (Sigma\u0026ndash;Aldrich, St. Louis, MO, USA) according to the manufacturer\u0026rsquo;s instructions. For each sample, 20 \u0026micro;g of total protein was separated on a 12.5% SDS-PAGE gel and transferred onto a nitrocellulose membrane (Millipore, Billerica, MA, USA). The membrane was then incubated with the following primary antibodies: rabbit anti-PRRX1 (Cat. No.:ZRB2165; Sigma-Aldrich), Recombinant anti-DDDDK tag (Binds to FLAG\u0026reg; tag sequence) antibody (Cat. No.: ab205606; Abcam), rabbit anti-PI3K antibody (Cat. No.: ab2958), rabbit anti-phosphorylated PI3K TYR458 antibody (Cat. No.: ab278545), rabbit anti-mTOR antibody (Cat. No.: ab134903), rabbit anti-phosphorylated mTOR at Ser2448 (Cat. No.: ab109268), rabbit anti-β-actin (Cat. No.: ab8227; Abcam, Cambridge, England), Rabbit anti-AKT antibody (Cat. No.: ab8805, Abcam) and Rabbit anti-phosphorylated AKT at Ser473 (Cat. No.: ab285140), at a dilution of 1:1000, and incubated overnight at 4\u0026deg;C. The membrane was then incubated with HRP-conjugated secondary antibody (Goat anti-rabbit IgG H\u0026amp;L antibody, Cat.No.: ab7090, Abcam, Cambridge, England) at a dilution of 1:5000 for 2 hours at room temperature. Immunodetection was performed using ECL reagents (Cat. No.: PRN2232; GE Healthcare Bio-Sciences).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Plasmid construction\u003c/h2\u003e\u003cp\u003eFor PRRX1 knockdown, the PRRX1 shRNA Plasmid (h) was purchased from Sigma-Aldrich: shScrambled (SHC016), shPRRX1 (mixture of TRCN0000020648 and TRCN0000020646).Overexpression plasmids (PRRX1A/PRRX1B/ANTXR1) were provided by Chengdu Rongyou Biotechnology Co., Ltd.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Cell viability assay\u003c/h2\u003e\u003cp\u003eCells were suspended and adjusted to a concentration of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/ml. 5000 cells/well were plated into a 96-well plate and incubated overnight. After 24 hours, the Cell Counting Kit-8 (CCK-8, Sigma\u0026ndash;Aldrich, St. Louis, MO, USA) solution was added and incubated for 4 hours at 37\u0026deg;C. The absorbance at 450 nm was measured using a microplate reader (Synergy 2 Multi-Mode Microplate Reader; BioTek, Winooski, VT, USA) to determine cell viability.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 CFSE assay\u003c/h2\u003e\u003cp\u003eCells were harvested and resuspended in serum-free PBS at a density of 1 \u0026times; 10^6 cells/mL. CFSE (Thermo Fisher Scientific, C34554) was added to the cell suspension at a final working concentration of 5 \u0026micro;M and incubated for 20 min at 37\u0026deg;C in the dark. The reaction was quenched by adding 5 volumes of complete culture medium and incubating for 5 min. The cells were then washed three times with PBS and seeded into culture plates. After cultivation for the indicated periods (1, 2, 3, 4, 5 Day). Observe the cell proliferation status through a fluorescence microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Plate Colony Formation Assay\u003c/h2\u003e\u003cp\u003eCells were trypsinized into a single-cell suspension and seeded into 6-well plates at a low density (500\u0026ndash;1000 cells per well). The cells were cultured in complete medium for 10\u0026ndash;14 days, with the medium changed every 3\u0026ndash;4 days. After formation of macroscopic colonies, the cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min, and then stained with 0.1% crystal violet (Beyotime, C0121) for 15 min at room temperature. The plates were rinsed with tap water and air-dried. Colonies containing more than 50 cells were photographed and counted manually.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Soft Agar Colony Formation Assay\u003c/h2\u003e\u003cp\u003eA base layer of 0.6% low-melting-point agarose (Invitrogen, 16520050) in complete medium was prepared in 6-well plates and allowed to solidify. A top layer of 0.3% agarose containing 5 \u0026times; 10^3 cells was plated on top of the base layer. After the top layer solidified, 1 mL of complete medium was added on top and replaced twice a week. The plates were incubated for 3\u0026ndash;4 weeks at 37\u0026deg;C. Colonies were stained with 0.005% Crystal Violet for 1 h and then imaged under a microscope. The number of colonies was quantified using ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Cell invasion assay using Transwell\u003c/h2\u003e\u003cp\u003eMatrigel (Millipore Corporation, Darmstadt, Germany) was diluted 1:2 in DMEM/F-12, and 60 \u0026micro;L of the diluted Matrigel was added to the upper chamber of the Transwell plates. The chambers were then incubated at 37\u0026deg;C in a 5% CO2 incubator for 2 hours. Single cells were resuspended in DMEM/F-12 and seeded into the upper chamber at a concentration of 2\u0026times;104 cells/well. Subsequently, 600 \u0026micro;L of DMEM was added to the lower chamber. After 24 hours of incubation, the inserts were collected, and the cells on the lower surface were fixed in 4% paraformaldehyde and stained with crystal violet for 15 minutes. The cells in five random fields were counted.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12 Immunofluorescence Staining\u003c/h2\u003e\u003cp\u003eCells grown on glass coverslips were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 5% BSA for 1 h at room temperature. The cells were then incubated with primary antibody (anti-ANTXR1, 1:400, Abcam, ab241067) overnight at 4\u0026deg;C. After washing with PBS, the cells were incubated with a fluorophore-conjugated secondary antibody (FITC, 1:500, Invitrogen, F-2765) and DAPI (1 \u0026micro;g/mL, Beyotime, C1002) for 1 h at room temperature in the dark. The coverslips were mounted onto glass slides with antifade mounting medium (Beyotime, P0126). Images were captured using a confocal laser scanning microscope (Zeiss LSM 880).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13 RT-qPCR\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from cells using TRIzol reagent (Invitrogen, 15596026) according to the manufacturer's protocol. cDNA was synthesized from 1 \u0026micro;g of total RNA using a HiScript II Q RT SuperMix kit (Vazyme, R223-01). Quantitative PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02) on a QuantStudio 5 Real-Time PCR System (Applied Biosystems). The relative mRNA expression levels were calculated using the 2^(-ΔΔCt) method and normalized to the expression of GAPDH as an internal control. The primer sequences used were as follows:PRRX1 F-5\u0026rsquo;-TGATGCTTTTGTGCGAGAA GA-3\u0026rsquo;;R-5\u0026rsquo;-AGGGAAGC GTTTTTATTGGCT-3\u0026rsquo;.ANTXR1 F-5\u0026prime;-ACAGTTGGCTCACAAATTCATCA-3\u0026prime;༛R-5\u0026prime;-TCACTG GCCCTTTCAAATCCT-3\u0026prime;.GAPDH F-5\u0026rsquo;-GCACCGTCAAGGCTGA GAAC-3\u0026rsquo;༛R-5\u0026rsquo;-TGGTGAAGACGCCAGTGGA-3\u0026rsquo;.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.14 Flow Cytometry for Cell Cycle Analysis\u003c/h2\u003e\u003cp\u003eCells were harvested, washed with PBS, and fixed in 70% ice-cold ethanol overnight at -20\u0026deg;C. The fixed cells were washed with PBS and then incubated with RNase A (100 \u0026micro;g/mL, Beyotime, ST577) and propidium iodide (PI, 50 \u0026micro;g/mL, Beyotime, ST511) for 30 min at 37\u0026deg;C in the dark. The DNA content of the stained cells was analyzed by flow cytometry (BD FACS Celesta). The cell cycle distribution (G0/G1, S, and G2/M phases) was determined using the ModFit LT software (v5.0).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.15 Statistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed using SPSS version 13.0. The data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD; x\u0026thinsp;\u0026plusmn;\u0026thinsp;s). The statistical significance of differences between groups was assessed using Student\u0026rsquo;s t-tests and one-way analysis of variance followed by Bonferroni post hoc analysis. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.1 In hepatocellular carcinoma, the expression level of PRRX1 is elevated.\u003c/h2\u003e\u003cp\u003eProtein proteomic analysis of liver cancer tissues and adjacent tissues revealed that the phosphorylation level of ANTXR1 in cancer tissues was significantly higher than that in adjacent tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, ANTXR1 expression showed no significant correlation with overall survival in HCC patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-A). Through analysis using the GEPIA online tool, we discovered that the transcriptional level of ANTXR1 was closely associated with PRRX1, and this association was notably stronger in cancer tissues compared to normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-B). Additionally, PRRX1 was found to significantly shorten the overall survival of liver cancer patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-C). PRRX1 has been shown to play a role in promoting malignant behaviors in multiple types of tumors, such as lung cancer. To explore the impact of PRRX1 on the development of liver cancer, we initially conducted IHC assays for PRRX1 on 175 pairs of hepatocellular carcinoma pathological specimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-D). The results indicated that PRRX1 was predominantly expressed in the cytoplasm, and the protein level of PRRX1 was significantly elevated in cancer tissues.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.2 The isoforms of PRRX1 promote the proliferation and malignant behavior of hepatocellular carcinoma cells.\u003c/h2\u003e\u003cp\u003eTo explore the function of PRRX1 in hepatocellular carcinoma cell lines, we employed RT-qPCR and Western Blot techniques to detect the mRNA and protein levels of PRRX1 in SNU449, SK-HEP-1, Hep3B, Huh-7, PLC-PRF-5, and RBE cell lines. The results indicated that although there were no substantial differences in the mRNA levels of PRRX1 among these cell lines, the protein levels of PRRX1 were remarkably higher in Huh-7 and SK-HEP-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-A, B). Subsequently, Huh- 7 and SK-HEP-1 cell lines were selected for subsequent experiments to investigate the tumor-regulatory functions of PRRX1. PRRX1 is a protein consisting of two subtypes, namely PRRX1A and PRRX1B. Previous reports have demonstrated that PRRX1A is involved in promoting the malignant phenotypes of lung cancer. To determine whether the function of PRRX1 in hepatocellular carcinoma is subtype-dependent, we respectively overexpressed FLAG-tagged PRRX1A and FLAG-tagged PRRX1B in Huh-7 and SK-HEP-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-C). The results of the CCK-8 assay showed that while the proliferation rates of cells transfected with either the vector or FLAG-PRRX1B did not exhibit significant alterations, the overexpression of FLAG-PRRX1A significantly accelerated cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-D). To further verify this, CFSE live-cell staining was performed, and cell proliferation was monitored under a fluorescence microscope. The results further confirmed that the overexpression of FLAG-PRRX1A, rather than FLAG-PRRX1B, promoted the proliferation of Huh-7 and SK-HEP-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-E, F). To further elucidate the impact of PRRX1A overexpression on tumor malignancy, we examined the effects of FLAG-PRRX1A overexpression on the migration and invasion capabilities of tumor cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-A), tumorigenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-B), and colony - forming ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-C). As anticipated, the overexpression of FLAG-PRRX1A significantly enhanced the malignant behaviors of tumor cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.3 PRRX1 transcriptionally upregulates ANTXR1.\u003c/h2\u003e\u003cp\u003eTo precisely define the interplay between PRRX1 and ANTXR1, we carried out Western blot analyses. The findings revealed that upon knockdown of PRRX1, the protein level of ANTXR1 was downregulated. Conversely, knockdown of ANTXR1 did not impact the protein level of PRRX1. Additionally, simultaneous overexpression of ANTXR1 during PRRX1 knockdown was able to reverse the decline in ANTXR1 protein levels induced by PRRX1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-A). To investigate whether PRRX1 influences the membrane localization of ANTXR1, we performed immunofluorescence experiments. The results demonstrated that shRNA-mediated knockdown of PRRX1 significantly decreased the membrane-localized ANTXR1 protein levels in both Huh-7 and SK-HEP-1 cells. This finding indicates that PRRX1 is essential for maintaining the stability of ANTXR1. It is noteworthy that in cells lacking PRRX1, ectopic overexpression of ANTXR1 fully restored its membrane localization. This suggests that PRRX1 regulates ANTXR1 at the post-transcriptional level, rather than being solely associated with its expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-B). This functional interaction implies that PRRX1 may modulate ANTXR1 through mechanisms such as protein stabilization, co-transport, or transcriptional activation pathways.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.4 PRRX1A, but not PRRX1B transcriptional upregulates ANTXR1 and its membrane location.\u003c/h2\u003e\u003cp\u003eTo elucidate the role of PRRX1 subtypes in modulating ANTXR1 transcription and membrane localization, we conducted RT-qPCR analyses following the overexpression of PRRX1A or PRRX1B in cells where PRRX1 had been knocked down. The results demonstrated that the overexpression of PRRX1A significantly upregulated the mRNA levels of ANTXR1, whereas PRRX1B did not exhibit such an effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e-A). This finding indicates that the homologous isoform PRRX1A specifically trans-activates ANTXR1. In line with this observation, confocal microscopy analysis showed that the overexpression of PRRX1A substantially enhanced the membrane-localized ANTXR1 protein levels in cells lacking PRRX1. In contrast, the overexpression of PRRX1B was unable to rescue the membrane localization of ANTXR1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e-B). Collectively, these data suggest that PRRX1A uniquely orchestrates the transcriptional activation and membrane trafficking of ANTXR1, thereby highlighting its pivotal functional role within the PRRX1-ANTXR1 axis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003e3. 5 PRRX1 activates the PI3K/Akt/mTOR signaling pathway by activating ANTXR1.\u003c/h3\u003e\n\u003cp\u003eCai and his colleagues reported that ANTXR1 serves as a crucial activator of the PI3K/AKT/mTOR signaling pathway, thereby facilitating cancer progression [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. We postulated that PRRX1 might activate the PI3K/AKT/mTOR signaling pathway through the upregulation of ANTXR1. In Huh-7 and SK-HEP-1 cells, interfering with either ANTXR1 or PRRX1 led to a suppression of the phosphorylation levels of PI3K/AKT/mTOR. Nevertheless, following the interference of PRRX1, the subsequent introduction of exogenous ANTXR1 reactivated the phosphorylation levels of PI3K/AKT/mTOR. This finding indicates that PRRX1 activates the PI3K/AKT/mTOR signaling pathway via the upregulation of ANTXR1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e-A).To further validate this mechanism, we added LY294002, a specific inhibitor of ANTXR1, to cells in which PRRX1 had been interfered with and then exogenous ANTXR1 had been introduced. The results demonstrated that the addition of LY294002 once again inhibited the phosphorylation levels of PI3K/AKT/mTOR (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e-B). Cell cycle analysis revealed that interfering with PRRX1 caused cell cycle arrest at the G1/G0 phase, and this effect could be reversed by the overexpression of ANTXR1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e-C). Furthermore, Western blot analysis results indicated that ANTXR1 may influence cell proliferation capacity by upregulating Cyclin D1 and P21 while downregulating P27 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e-D).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur study demonstrates that PRRX1 is significantly upregulated in HCC tissues and transcriptionally regulates ANTXR1. Functional assays revealed that the PRRX1A isoform, but not PRRX1B, exerts tumor-promoting effects in HCC cells. Although we identified that PRRX1 promotes HCC malignancy via ANTXR1, the precise mechanistic details of this regulation warrant further investigation.\u003c/p\u003e\u003cp\u003ePRRX1 (Paired Related Homeobox 1) is a transcription factor that plays important biological functions in embryonic development and cell differentiation. PRRX1 plays a crucial role in embryonic development, contributing to the formation of various organs and tissues such as the heart, kidneys, lungs, and skeletal system. PRRX1 promotes the normal formation and development of tissues and organs by regulating gene expression and participating in cell fate determination and cell migration. PRRX1 is involved in the regulation of cell differentiation, transforming cells from a primitive state into specific cell types. PRRX1 also has an impact on the maintenance and differentiation of embryonic stem cells, which are cells with self-renewal and differentiation potential. PRRX1\u0026rsquo;s role in tumorigenesis and tumor development is completely different. The expression and function of PRRX1 in breast cancer have received extensive attention. Studies have shown that PRRX1 may be involved in the proliferation, invasion, and metastasis of breast cancer cells. It may serve as a potential therapeutic target or diagnostic marker for breast cancer. However, the specific mechanisms and regulatory networks of PRRX1 still require further research for a comprehensive understanding.\u003c/p\u003e\u003cp\u003eIsoforms are protein variants that arise from the same gene with slight differences. They may exhibit variations in structure, function, and expression. PRRX1 has two isoforms, PRRX1A and PRRX1B, which display significant functional differences. PRRX1A and PRRX1B are involved in regulating the processes of epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], which modulate metastasis in hepatocarcinomas (29). This isoform-specific regulation of the EMT-MET axis has been revealed in breast (30), colorectal (31, 32), and gastric cancer (33). In our results, PRRX1A exerts promoting effects on proliferation, invasion, colony formation and tumor formation in soft agar, but not PRRX1B, in hepatocarcinoma cells. We also evaluated the transcriptional regulation of PRRX1 on ANTXR1. However, it is still unclear that whether the regulation is dependent on different isoforms, which is worth to be investigated in further study.\u003c/p\u003e\u003cp\u003eANTXR1 (Anthrax Toxin Receptor 1), also known as TEM8 (Tumor endothelial marker 8), is a transmembrane protein closely involved in the regulation of multiple signaling pathways and cellular functions. ANTXR1 interacts with key proteins in the mTOR/PI3K signaling pathway. For example, ANTXR1 can form a complex with PI3K (Phosphoinositide 3-kinase) and promote the activation of the PI3K signaling pathway. This interaction may be achieved through specific domains within the cytoplasmic region of ANTXR1. Furthermore, the activation of ANTXR1 can influence processes such as cell proliferation, survival, and metabolism through the PI3K/AKT/mTOR signaling pathway. The interaction between ANTXR1 and PI3K activates PI3K, subsequently activating downstream proteins such as AKT (Protein Kinase B) and mTOR (Mammalian Target of Rapamycin), promoting cell proliferation and growth. In the context of cancer, abnormal activation of the ANTXR1-mTOR/PI3K signaling pathway plays a crucial role in the development of various types of cancers. High expression of ANTXR1 is associated with cancer cell growth, invasion, and metastasis, potentially promoting tumor cell proliferation and survival through the PI3K/AKT/mTOR signaling pathway.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn our experiments, we observed that interference with PRRX1 and ANTXR1 can inhibit the mTOR/PI3K signaling pathway. This inhibition occurs by suppressing the phosphorylation of key proteins, indicating the essential role of PRRX1/ANTXR1 in the activity of mTOR/PI3K and subsequently affecting cell proliferation and the cell cycle progression. In conclusion, we speculate that PRRX1 likely exerts as a tumor promoter in hepatocarcinoma. However, further study is required to clarify the mechanism.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eQ.Y.H. and Y.K.W. conceived and designed the study. F.W. and Q.L. performed the cellular experiments. H.Q.C. and T.W. conducted the animal experiments. Q.Y.H. and Y.K.W. wrote and revised the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eEthics Approval and Consent to Participate\u003c/p\u003e\n\u003cp\u003eThe human tissue-related experiments in this study were approved by the Ethics Committee of Chengdu University of Traditional Chinese Medicine (Approval No. 2025016) and were conducted in strict accordance with the Declaration of Helsinki. As this study was a retrospective analysis utilizing anonymized samples, the requirement for informed consent was waived by the Chengdu University of Traditional Chinese Medicine Ethics Committee.\u003c/p\u003e\n\u003cp\u003eAcknowledgment\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Professor Huimin Shi (Peking University, Beijing, China) for language editing.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study was supported by National Natural Science Foundation of China (NSFC, 81973684), 2023NSFSC1760 and Xinglin scholar (QJRC2022007)\u003c/p\u003e\n\u003cp\u003eData access statement\u003c/p\u003e\n\u003cp\u003eThe data analyzed in FIG1 is obtained from the PRIDE database(PXD047213, https://www.ebi.ac.uk/pride/archive/projects/PXD047213). The data analyzed in FIG2-A-C is from the TCGA database and was generated through GEPIA (http://gepia.cancer-pku.cn/).The other data generated in this study are available upon request from the corresponding author.\u003c/p\u003e\n\u003cp\u003eConflict of Interest\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eAll authors reviewed and approved the final manuscript. Qiongying Hu is responsible for the overall content as the guarantor.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eShimozaki K, Clemenson GD, Jr., Gage FH. Paired related homeobox protein 1 is a regulator of stemness in adult neural stem/progenitor cells. J. Neurosci. 2013;33:4066\u0026ndash;4075.\u003c/li\u003e\n\u003cli\u003eIhida-Stansbury K, et al. Paired-related homeobox gene Prx1 is required for pulmonary vascular development. Circ. Res. 2004;94:1507\u0026ndash;1514. \u003c/li\u003e\n\u003cli\u003eOcana OH, et al. Metastatic colonization requires the repression of the epithelial-mesenchymal transition inducer PRRX1. Cancer Cell. 2012;22:709\u0026ndash;724. \u003c/li\u003e\n\u003cli\u003eGuo J, et al. PRRX1 promotes epithelial-mesenchymal transition through the Wnt/\u0026beta;-catenin pathway in gastric cancer. Med. Oncol.2015;32:393.\u003c/li\u003e\n\u003cli\u003eTakahashi Y, et al. Paired related homoeobox 1, a new EMT inducer, is involved in metastasis and poor prognosis in colorectal cancer. Brit J. Cancer. 2013;109:307\u0026ndash;311. \u003c/li\u003e\n\u003cli\u003eMarchand B, et al. PRRX1 isoforms cooperate with FOXM1 to regulate the DNA damage response in pancreatic cancer cells. Oncogene.2019;38:4325\u0026ndash;4339.\u003c/li\u003e\n\u003cli\u003eLi Y, et al. Paired related homeobox 1 transactivates dopamine D2 receptor to maintain propagation and tumorigenicity of glioma-initiating cells. J. Mol. Cell Biol. 2017;9:302\u0026ndash;314.\u003c/li\u003e\n\u003cli\u003eOcana OH, et al. Metastatic colonization requires the repression of the epithelial-mesenchymal transition inducer PRRX1. Cancer Cell. 2012;22:709\u0026ndash;724. \u003c/li\u003e\n\u003cli\u003eFan M, et al. Downregulation of PRRX1 via the p53-dependent signaling pathway predicts poor prognosis in hepatocellular carcinoma. Oncol. Rep. 2017;38:1083\u0026ndash;1090. \u003c/li\u003e\n\u003cli\u003eHardin H, et al. The roles of the epithelial-mesenchymal transition marker PRRX1 and miR-146b-5p in papillary thyroid carcinoma progression. Am. J. Pathol. 2014;184:2342\u0026ndash;2354. \u003c/li\u003e\n\u003cli\u003eTorre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65(2):87\u0026ndash;108. \u003c/li\u003e\n\u003cli\u003eLiang XH, Loncarevic IF, Tang ZY, Yu YQ, Zentgraf H, Schroder CH. Resection of hepatocellular carcinoma: oligocentric origin of recurrent and multinodular tumours. J Gastroenterol Hepatol. 1991;6(1):77\u0026ndash;80. \u003c/li\u003e\n\u003cli\u003eChambers AF, Groom AC, MacDonald IC. Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer. 2002;2(8):563\u0026ndash;572. \u003c/li\u003e\n\u003cli\u003eYang M. Y., Chaudhary A., Seaman S., Dunty J., Stevens J., Elzarrad M. K., et al. (2011). The cell surface structure of tumor endothelial marker 8 (tem8) is regulated by the actin cytoskeleton. Biochim. Biophys. Acta 1813, 39\u0026ndash;49. \u003c/li\u003e\n\u003cli\u003eOpoku-Darko M., Yuen C., Grassi C., Gratton K., Bathe O. (2007). P54. J. Surg. Res. 137, 260. \u003c/li\u003e\n\u003cli\u003eChaudhary A., Hilton M. B., Seaman S., Haines D. C., Stevenson S., Lemotte P. K., et al. (2012). Tem8/antxr1 blockade inhibits pathological angiogenesis and potentiates tumoricidal responses against multiple cancer types. Cancer Cell 21, 212\u0026ndash;226.\u003c/li\u003e\n\u003cli\u003eXu J., Yang X., Deng Q., Yang C., Wang D., Jiang G., et al. (2021). Tem8 marks neovasculogenic tumor-initiating cells in triple-negative breast cancer. Nat. Commun. 12, 4413.\u003c/li\u003e\n\u003cli\u003eLi M., Fang L., Kwantwi L. B., He G., Luo W., Yang L., et al. (2021). N-myc promotes angiogenesis and therapeutic resistance of prostate cancer by tem8. Med. Oncol. 38, 127. \u003c/li\u003e\n\u003cli\u003eSun K-R., Lv H-F., Chen B-B., Nie C. Y., Zhao J., Chen X. B. (2021). Latest therapeutic target for gastric cancer: Anthrax toxin receptor 1. World J. Gastrointest. Oncol. 13, 216\u0026ndash;222. \u003c/li\u003e\n\u003cli\u003eAlcal\u0026aacute; S., Martinelli P., Hermann P. C., Heeschen C., Sainz B. (2019). The anthrax toxin receptor 1 (antxr1) is enriched in pancreatic cancer stem cells derived from primary tumor cultures. Stem Cells Int. 2019, 1378639.\u003c/li\u003e\n\u003cli\u003eKusaba Y., Kajihara I., Sakamoto R., Maeda-Otsuka S., Yamada-Kanazawa S., Sawamura S., et al. (2021). Overexpression of tumor endothelial marker 8 protein predicts poor prognosis in angiosarcoma. J. Dermatol. 48, E514\u0026ndash;E516. \u003c/li\u003e\n\u003cli\u003eŁ Pietrzyk, Korolczuk A., Matysek M., Arciszewski M. B., Torres K. (2021). Clinical value of detecting tumor endothelial marker 8 (antxr1) as a biomarker in the diagnosis and prognosis of colorectal cancer. Cancer Manag. Res. 13, 3113\u0026ndash;3122.\u003c/li\u003e\n\u003cli\u003eGong Q., Deng J., Zhang L., Zhou C., Fu C., Wang X., et al. (2021). Targeted silencing of tem8 suppresses non-small cell lung cancer tumor growth via the erk/bcl-2 signaling pathway. Mol. Med. Rep. 24, 595.\u003c/li\u003e\n\u003cli\u003eFern\u0026aacute;ndez-Cort\u0026eacute;s M., Delgado-Bellido D., Oliver F. J. (2019). Vasculogenic mimicry: Become an endothelial cell \u0026quot;but not so much. Front. Oncol. 9, 803.\u003c/li\u003e\n\u003cli\u003eChen D., Bhat-Nakshatri P., Goswami C., Badve S., Nakshatri H. (2013). Antxr1, a stem cell-enriched functional biomarker, connects collagen signaling to cancer stem-like cells and metastasis in breast cancer. Cancer Res. 73, 5821\u0026ndash;5833. \u003c/li\u003e\n\u003cli\u003eCai C, Dang W, Liu S, et al. Anthrax toxin receptor 1/tumor endothelial marker 8 promotes gastric cancer progression through activation of the PI3K/AKT/mTOR signaling pathway. Cancer Sci. 2020;111(4):1132-1145. \u003c/li\u003e\n\u003cli\u003eMarchand B, Pitarresi JR, Reichert M, et al. PRRX1 isoforms cooperate with FOXM1 to regulate the DNA damage response in pancreatic cancer cells. Oncogene 2019;38:4325-39.\u003c/li\u003e\n\u003cli\u003eOca\u0026ntilde;a OH, Coskun H, Minguill\u0026oacute;n C, et al. A right-handed signalling pathway drives heart looping in vertebrates. Nature 2017;549:86-90.\u003c/li\u003e\n\u003cli\u003eTakano S, Reichert M, Bakir B, et al. PRRX1 isoform switching regulates pancreatic cancer invasion and metastatic colonization. Genes Dev 2016;30:233-47.\u003c/li\u003e\n\u003cli\u003eOca\u0026ntilde;a OH, C\u0026oacute;rcoles R, Fabra A, et al. Metastatic colonization requires the repression of the epithelial-mesenchymal transition inducer PRRX1. Cancer Cell 2012;22:709-24.\u003c/li\u003e\n\u003cli\u003eTakahashi Y, Sawada G, Kurashige J, et al. Paired related homoeobox 1, a new EMT inducer, is involved in metastasis and poor prognosis in colorectal cancer. Br J Cancer 2013;109:307-11.\u003c/li\u003e\n\u003cli\u003eZheng L, Zhang Y, Lin S, et al. Down-regualtion of miR-106b induces epithelial-mesenchymal transition but suppresses metastatic colonization by targeting PRRX1 in colorectal cancer. Int J Clin Exp Pathol 2015;8:10534-44.\u003c/li\u003e\n\u003cli\u003eEllsworth RE, Blackburn HL, Shriver CD, et al. Molecular heterogeneity in breast cancer: State of the science and implications for patient care. Semin Cell Dev Biol 2017;64:65-72.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"PRRX1, isoform, ANTXR1, hepatocarcinoma, mTOR/PI3K signaling","lastPublishedDoi":"10.21203/rs.3.rs-7814012/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7814012/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProteomic analysis of hepatocellular carcinoma (HCC) and paired adjacent non-tumor tissues revealed a significant increase in ANTXR1 phosphorylation in HCC samples. Using the GEPIA online tool, we identified a strong positive correlation between ANTXR1 and PRRX1 in HCC tissues. Moreover, the transcriptional level of PRRX1 was closely associated with overall survival in HCC patients. PRRX1, a homeodomain transcription factor, exists in two subtypes: PRRX1A and PRRX1B. These subtypes have been implicated in several malignancies, including pancreatic, breast, and lung cancers; however, their roles in HCC remain unclear.\u003c/p\u003e\u003cp\u003eImmunohistochemical (IHC) analysis of 175 paired clinical samples demonstrated significant PRRX1 overexpression in HCC tissues compared to adjacent tissues. IIndividual overexpression of PRRX1 isoforms demonstrated that PRRX1A, but not PRRX1B, markedly promoted hepatocellular carcinoma cell proliferation.\u003c/p\u003e\u003cp\u003eFurthermore, knockdown of PRRX1 resulted in significant downregulation of ANTXR1 protein levels in both Huh-7 and SK-HEP-1 cell lines, suggesting that PRRX1 positively regulates ANTXR1. Notably, only PRRX1A overexpression rescued the suppressed ANTXR1 transcription and altered its subcellular localization following PRRX1 knockdown.\u003c/p\u003e\u003cp\u003eMechanistic investigations revealed that both PRRX1 and ANTXR1 contribute to tumor progression through activation of the PI3K/Akt/mTOR signaling pathway. In summary, our results indicate that PRRX1, particularly the PRRX1A subtype, acts as a critical promoter in hepatocellular carcinoma progression.\u003c/p\u003e","manuscriptTitle":"PRRX1 regulates malignancies partially via regulating ANTXR1 and thus activating PI3K/mTOR signaling in hepatocarcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-08 07:33:44","doi":"10.21203/rs.3.rs-7814012/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"79445436-8fa2-4c61-9ebe-e6833296ec0b","owner":[],"postedDate":"December 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-11T12:42:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-08 07:33:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7814012","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7814012","identity":"rs-7814012","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}