ESRRA-Driven PHF5A Activation Promotes Hepatocellular Carcinoma Progression and is Therapeutically Targeted by Rosuvastatin

ESRRA-Driven PHF5A Activation Promotes Hepatocellular Carcinoma Progression and is Therapeutically Targeted by Rosuvastatin | 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 ESRRA-Driven PHF5A Activation Promotes Hepatocellular Carcinoma Progression and is Therapeutically Targeted by Rosuvastatin Xiang Cai, Zhiping Wan, Shuying Huang, Yutian Chong, Yusheng Jie, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7867831/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 Background The estrogen related receptor alpha (ESRRA), a key member of the estrogen receptor-related receptor (ERR) family, has been extensively implicated in tumor progression across multiple cancers. Existing studies highlight its pivotal role in cancer cell proliferation and migration. However, its specific function and underlying molecular mechanisms in hepatocellular carcinoma (HCC) remain incompletely understood. Methods The effects of ESRRA on HCC cells proliferation and migration were investigated in vitro (CCK-8, colony formation, EdU proliferation, wound-healing, transwell assays, and Epithelial-mesenchymal transition (EMT) marker analysis) and in vivo (Balb/c nude mouse subcutaneous xenograft and lung metastasis models). RNA sequencing and dual-luciferase reporter assays were employed to identify ESRRA’s downstream targets and pathways. Results ESRRA promoted HCC cell proliferation and migration in vitro and in vivo . Mechanistically, ESRRA transcriptionally upregulated PHD finger protein 5A (PHF5A), which subsequently activated the PI3K/AKT signaling cascade. The cholesterol-lowering drug rosuvastatin exerted anti-tumor effects on HCC by downregulating ESRRA and, meanwhile, suppressing its transcriptional activity through depleting intracellular cholesterol. Conclusion ESRRA promotes HCC progression via the PHF5A /PI3K/AKT axis and mediates rosuvastatin's anti-tumor effect. Targeting ESRRA-PHF5A may be a therapeutic strategy for HCC. ESRRA PHF5A hepatocellular carcinoma statin cholesterol Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Liver cancer exhibits a concerning global health burden, ranking as the sixth most prevalent malignancy and the third leading cause of cancer-related deaths worldwide, imposing substantial socioeconomic and healthcare challenges[ 1 ]. Among primary liver cancers, hepatocellular carcinoma (HCC) is the most prevalent histological subtype. Therefore, elucidating the molecular mechanisms underlying HCC progression, identifying innovative therapeutic strategies, and optimizing existing treatment modalities are imperative. The superfamily of nuclear receptors (NRs), which consists of transcriptional factors, has been extensively investigated regarding their functions in the development and progression of cancer [ 2 , 3 ]. Additionally, NRs possess unique ligand-binding sites that can interact with small molecules. This characteristic has rendered them appealing targets for the development of cancer treatments [ 3 ]. The roles of NRs in HCC vary. Some promote HCC progression, such as Nuclear Receptor Subfamily 4 Group A Member 3 (NR4A3) and Nuclear Receptor Subfamily 2 Group E (e.g., HNF4α), while others suppress it, like Nuclear Receptor Subfamily 0, Group B, Member 1 (NR0B1)[ 4 – 6 ]. The estrogen receptor (ER)-related receptors (ERRs), including ESRRA, ESRRB, and ESRRG, constitute the NR3B subgroup of NRs and play pivotal roles in regulating cellular metabolism and development[ 7 ]. Among these, ESRRA is predominantly expressed in tissues with high energy demands, while ESRRB is active during early embryonic development, and ESRRG is primarily found in the central nervous system and spinal cord [ 3 ]. Notably, ESRRA directly regulates key metabolic pathways, such as mitochondrial biogenesis, oxidative phosphorylation, and glycolysis, which were required for the proliferation and migration of cancer cells[ 8 , 9 ]. Given that cancer cells similarly exhibit heightened energy requirements, it is unsurprising that ESRRA—more than other ERRs—has been extensively linked to various cancers, including breast, ovarian, prostate, and esophageal cancers [ 3 , 10 – 12 ]. However, despite its well-established role in other cancers, the role of ESRRA in HCC remains underexplored. Consequently, the present study aims to elucidate the precise role of ESRRA in the development of HCC and the underlying molecular mechanisms. Moreover, it may help to identify novel and potentially effective targets for the treatment of HCC. Materials and Methods Cell culture The human normal liver epithelial cell line (THLE2) and hepatocellular carcinoma (HCC) cell lines (HepG2, MHCC-97H, Huh7, and SNU-449) were obtained from the Cell Bank of the Chinese Academy of Sciences. All cell lines were authenticated by short tandem repeat (STR) profiling prior to experimental use. THLE2, HepG2, and MHCC-97H cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS; Procell, Wuhan, China). SNU-449 cells were maintained in RPMI-1640 medium (Gibco, CA, USA) containing 10% FBS (Procell). All cells were incubated at 37°C in a humidified atmosphere with 5% CO2. siRNA and plasmid construction siRNAs targeting ESRRA and PHD finger protein 5A (PHF5A), along with a negative control siRNA, were synthesized by Hanyi Biotechnology (Shanghai, China) (sequences provided in Supplementary Table 1). For overexpression studies, the pcDNA3.1-ESRRA and pcDNA3.1-PHF5A plasmids, as well as the empty vector control (pcDNA3.1), were obtained from Genepharma (Shanghai, China) (plasmid sequences detailed in Supplementary Tables 2 and 3). Transfections were performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Briefly, 50 nM siRNA or 2 µg plasmid DNA was diluted in Opti-MEM, mixed with Lipofectamine 3000 reagent (1:1 ratio, v/v), and incubated for 15 min at room temperature (RT) before being added to the cells. The knockdown efficiency (for siRNA) and overexpression efficiency (for plasmids) were confirmed by Western blot analysis. Protein Extraction and Western Blot Analysis Protein Extraction and Western Blot Analysis Total protein was extracted using RIPA lysis buffer supplemented with protease inhibitors. The lysates were then separated by SDS-PAGE and electrophoretically transferred onto PVDF membranes. To minimize nonspecific binding, the membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature. Subsequently, the membranes were incubated with primary antibodies overnight at 4°C, followed by 1 h incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature. Protein signals were visualized using an enhanced chemiluminescence (ECL) detection system (ZETA LIFE). Detailed information on the antibodies used is provided in Supplementary Table 4.\ Colony formation assay HCC cells were digested by trypsin, counted, and seeded at a low density (typically 500–1000 cells per well) in 6-well plates containing complete growth medium. The plates were then incubated at 37°C in a humidified atmosphere with 5% CO₂ for two weeks, allowing colony formation. After that, cells were gently washed with phosphate-buffered saline (PBS) for 3 times, fixed with 4% paraformaldehyde for 20 min, and stained with crystal violet for 30 min at room temperature. Excess stain was removed by washing with distilled water, and plates were air-dried. Colonies consisting of more than 50 cells were counted. Cell Counting Kit-8 (CCK-8) Assay HCC cells were seeded in 96-well plates at a density of 3,000–5,000 cells/well in 100 µL of complete culture medium and allowed to adhere. At days 0, 1, 2, and 3, 10 µL of CCK-8 solution was added to each well, followed by incubation at 37°C for 2 h. Absorbance was measured at 450 nm using a Tecan SPARK microplate reader (Switzerland). EdU proliferation assay HCC cells were seeded in 48-well plates (or chamber slides) in complete growth medium. After 48 h of culture, cells were incubated with EdU solution (50 µM, BeyoClick™ EdU Kit, Beyotime, Shanghai, China) for 2–4 h. Subsequently, cells were washed by PBS, and fixed with 4% paraformaldehyde for 20 min. Permeabilization was performed using 0.5% Triton X-100 in PBS for 30 min. The Click-iT® reaction cocktail was prepared according to the manufacturer’s protocol. Cells were incubated with the reaction mixture for 30 min (protected from light). Cells were washed and counterstained with Hoechst 33342 (1 µg/mL, 10 min, RT) for nuclear visualization. Fluorescence images were captured using a Zeiss inverted fluorescence microscope at 20× magnification. Wound-healing assay HCC cells were seeded in 6-well plates at a density of 5 × 10⁵ cells/well in complete growth medium until reaching 90–100% confluence. A sterile 20 µL pipette tip was used to create a straight scratch wound in the cell monolayer. After that, the cells were gently washed with PBS to remove detached cells and incubated with 2% FBS-supplemented medium. Images were captured after 24 h using a Zeiss inverted fluorescence microscope (10×). Wound healing magnitude was quantified by measuring the relative wound closure compared with control cells at 24 hours[ 13 ]. Transwell migration assay HCC cells were harvested and resuspended in serum-free medium. Polycarbonate membrane inserts (8-µm pore size) were placed in 24-well plates. The lower chamber was filled with 600 µL of chemoattractant medium (DMEM with 10% FBS). 100 µL of cell suspension was added to the upper chamber. Cells were incubated at 37°C for 24 hours. Non-migrated cells on the upper membrane surface were removed. Migrated cells on the lower membrane surface were fixed with 4% paraformaldehyde and stained with crystal violet for 15 min. Migrated cells were counted under a Zeiss inverted fluorescence microscope at 20× fields. Cell cycle analysis HCC cells were harvested, washed twice with ice-cold PBS, and fixed in 70% ice-cold ethanol at 4°C overnight. Fixed cells were resuspended in PBS and treated with RNase A at 37°C for 30 min. Afterwards, cells were stained with propidium iodide (Solarbio, Beijing, China) in the dark for 30 min. Finally, the cell cycle distributions of the samples were detected by flow cytometry (Cytek Biosciences, Fremont, CA). The cell cycle distribution was analyzed using the software FlowJo_v10.9.0 (Oregon, USA). Animal experiments Animal experiments were conducted with strict adherence to ethical and scientific standards. BALB/c nude male mice aged 3 weeks were obtained from GemPharmatech Co., Ltd (Nanjing, China). All procedures involved in the animal experiments were approved by the Committee of Animal Experimental Ethics (IACUC − 202505166). The mice were housed in specific pathogen - free (SPF) facilities under well - controlled conditions, including a 12 - h light/dark cycle, a temperature of 22 ± 2°C, and a humidity of 50 ± 10%, with unrestricted access to autoclaved food and water. To establish subcutaneous tumor models in nude mice, MHCC-97H and Huh7 cells were seeded in 10-cm culture dishes. The Huh7 cells were infected with either the negative lentivirus or the lentivirus-based ESRRA overexpression vector from Jima Company (Shanghai, China). The sequence of the ESRRA lentivirus is detailed in Supplementary Table 5. Subsequently, selection was carried out in a medium containing puromycin (2µg/ml) from Jima Company (Shanghai, China) to obtain stable transfectants. When the cells reached 80–90% confluency, the MHCC − 97H and Huh7 cells were harvested using 0.25% trypsin-EDTA and resuspended. After a 7-day acclimatization period, 5×10⁶ HCC cells in a 100µL suspension were subcutaneously injected into the right flank of the BALB/c nude male mice. The mice were monitored daily for tumor growth and overall health status. Ten days after the subcutaneous injection of MHCC-97H cells, the tumor - bearing mice were randomly divided into three groups: the XCT790 group, which received an intraperitoneal injection of 8 mg/kg XCT790 dissolved in dimethyl sulfoxide (DMSO); the vehicle control group, which was given an equivalent volume of DMSO; and the blank control group, which received an equivalent volume of PBS[ 14 ]. Tumor volume was measured every 3–4 days using calipers and calculated using the formula: tumor volume = (length × width²)/2. For the Huh7 xenografts, the tumors were removed 4 weeks after injection. For the MHCC-97H models, the tumors were harvested 3 weeks after XCT790 treatment. Prior to tumor collection, the mice were euthanized by CO₂ asphyxiation. For the lung metastasis model, MHCC-97H cells and Huh7 cells (from both empty vector and ESRRA overexpression groups) were collected and injected via the tail vein into nude mice (2×10⁶ cells per mouse). Two weeks later, mice injected with MHCC-97H cells were divided into two groups: XCT790 group (8 mg/kg) and DMSO group (equivalent volume of vehicle), with treatments administered via intraperitoneal injection. Five weeks after tail vein injection, all mice (injected with either MHCC-97H or Huh7 cells) were euthanized by CO₂ asphyxiation, and lung tissues were isolated for subsequent hematoxylin-eosin (HE) staining. Immunohistochemistry (IHC) analysis Subcutaneous tumor tissues were fixed in neutral buffered formalin for 24 h at room temperature, followed by dehydration through a graded ethanol series. Tissues were cleared in xylene, infiltrated with paraffin, and embedded in paraffin blocks. The paraffin-embedded tumor tissues were cut into 4-µm-thick sections, dewaxed with xylene and rehydrated with descending ethanol. After antigen retrieval, the tissue slides were blocked with 5% bovine serum albumin (BSA) at room temperature and then Incubated overnight at 4°C with the primary antibodies anti-ki67, anti-ESRRA, and anti-Plant Homeodomain Finger Protein 5A (PHF5A). The detailed information of antibodies was listed in Supplementary Table 6. HE staining Upon euthanasia, lung tissues from each group of nude mice were fixed in 10% formalin for 48 hours, paraffin-embedded, and sectioned at 4 µm thickness. The sections were mounted on glass slides, dewaxed in xylene, and rehydrated through a graded ethanol series. For histological staining, sections were treated with hematoxylin for 2 minutes at room temperature, rinsed under running water for 5 minutes to restore a blue hue, and counterstained with eosin for 5 minutes. Finally, slides were dehydrated through an ascending ethanol series, cleared in xylene, and prepared for analysis. RNA sequencing RNA sequencing and data analysis were performed according to our previously published research and was performed by Hangzhou Lianchuan Bio Technologies Co[ 15 – 17 ]. Briefly, total RNA was extracted by TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Each sample in normal control (NC) group and ESRRA siRNA group were resultant mix of nine RNA extraction. The total RNA quantity and purity were analyzed with a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA). RNA fragments are enriched by oligo (dT) magnetic beads (Dynabeads Oligo (dT), Thermo Fisher, USA). Finally, RNA sequencing was performed using Illumina Novaseq™ 6000 platform. Quantitative real-time PCR For quantitative real - time PCR analysis, total RNA was isolated from the cultured cell lines using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Subsequently, complementary DNA (cDNA) was synthesized with a commercial reverse transcription kit according to the manufacturer’s instructions. (Takara, Tokyo, Japan). After cDNA synthesis, quantitative PCR amplification was carried out using a Light Cycler 480II Real-Time PCR System (Roche Diagnostics, Mannheim, Germany). This was achieved by using SYBR Green Master Mix (TAKARA) on the LightCycler 480 instrument (Roche, Basel, Switzerland). The sequences of all PCR primers employed in this experiment are detailed in Supplementary Table 7. Dual luciferase reporter assay The binding sites of ESRRA at the PHF5A promoter were predicted using the JASPAR database ( https://jaspar.genereg.net/ ). Luciferase reporter assays were performed following previously published protocols[ 18 , 19 ]. The wild-type or mutant promoters of human PHF5A were transduced into pGL3-basic vectors (Supplementary table 8–12). The ESRRA expression plasmid or empty vector, PGC1-α expression plasmid, and wild-type or mutant pGL3-basic vectors were co-transfected into HCC cells. To assess the transcriptional activity of ESRRA, the ESRRA expression plasmid or an empty vector was co-transfected with the ERRE-luc reporter (Yeasen, Shanghai, China) into HCC cells. These HCC cells were pre-treated under three distinct conditions: with rosuvastatin (25 µM, MCE, New Jersey, USA), with a combination of rosuvastatin and mevalonate (3 mM, Sigma-Aldrich, Saint Louis, USA), or with an equal volume of DMSO[ 20 ]. Luciferase and renilla assays were conducted using a dual-luciferase reporter system (Transgene, Beijing, China) according to the manufacturer’s protocol. The Firefly and renilla luciferase signals were detected using a microplate reader (Tecan SPARK, Switzerland). Time-course analysis Gene expression data of human primary hepatocyte treated with rosuvastatin for 24 hours or 48 hours were obtained from dataset GSE24188 for time-course analysis. The R (R Core Team 2021) package (mfuzz) (Futschik and Carlisle 2005) to perform time-course analysis. Statistical analysis All quantitative assays were conducted in triplicate. Mfuzz ( http://mfuzz##sysbiolab##eu ) was used for time course analysis. Statistical analyses and graphical representation were performed using GraphPad Prism 8.0.1 (GraphPad Software, San Diego, CA, USA). Results are presented as mean ± standard deviation, with P < 0.05 considered statistically significant. Results ESRRA is upregulated in HCC and facilitates cell proliferation in vitro To investigate the potential role of ESRRA in HCC, we firstly explored the expression level of ESRRA in human HCC tissues using data from TGCA database. Analysis of the TCGA database revealed significantly higher ESRRA expression in human HCC tissues compared to normal liver tissues (Fig. 1 a). Consistently, ESRRA levels were markedly increased in multiple HCC cell lines relative to immortalized normal human hepatocytes (THLE2) (Fig. 1 b, Supplementary Fig. 1a). To investigate the functional role of ESRRA, we established ESRRA-knockdown HCC cell models and confirmed the knockdown efficiency at the protein level via western blotting (Supplementary Fig. 1b-c). Functional assays demonstrated that ESRRA silencing significantly inhibited HCC cell proliferation, as evidenced by CCK-8 assays, EdU proliferation assays, and colony formation assays (Fig. 1 c-h, Supplementary Fig. 1d-e). Moreover, ESRRA knockdown induced G1 phase arrest, further supporting its role in regulating cell proliferation (Fig. 1 i-j). Conversely, ESRRA overexpression (validated by Western blot; Supplementary Fig. 1f–g) promoted HCC cell proliferation as confirmed by colony formation assays, EdU proliferation assays, and CCK-8, (Fig. 1 k-p, Supplementary Fig. 1h-i). ESRRA overexpression also facilitated G1/S transition (Fig. 1 q-s). The above findings underscore the impact of ESRRA in promoting proliferation in HCC cells. ESRRA accelerates HCC cell migration in vitro To evaluate the effect of ESRRA on HCC cell migration, we first performed wound-healing assays in ESRRA-knockdown HCC cells. Silencing ESRRA significantly impaired migratory capacity, as evidenced by reduced wound closure (Fig. 2 a–c) and fewer invading cells in transwell assays (Fig. 2 d). Given the pivotal role of epithelial-mesenchymal transition (EMT) in cancer metastasis[ 21 , 22 ], we performed western blot analysis to detect the expression of EMT markers in HCC cells treated with ESRRA siRNA. Western blotting revealed that ESRRA knockdown downregulated mesenchymal markers (N-cadherin, vimentin) while upregulating the epithelial marker E-cadherin (Fig. 2 e). Conversely, overexpression of ESRRA significantly enhanced the migratory ability of HCC cells, as evidenced by wound-healing assays and transwell experiments (Fig. 2 f-h). Concurrently, Western blot analysis revealed that ESRRA overexpression promoted the EMT process: it increased the expression of mesenchymal markers (N-cadherin and vimentin) and decreased the expression of the epithelial marker E-cadherin (Fig. 2 i). These data establish ESRRA as a critical driver of HCC migration and EMT. ESRRA promotes tumor growth and metastasis in vivo To further confirm the role of ESRRA in the tumor growth and metastasis of HCC, subcutaneous tumor growth models were established in nude mice. Compared to the blank control and empty vector groups, ESRRA overexpression significantly enhanced tumor growth, as evidenced by increased tumor volume and weight (Fig. 3 a-c). No significant difference in body weight was observed across the three groups (Fig. 3 d). Consistently, pharmacological inhibition of ESRRA using XCT790 (an ESRRA antagonist) markedly suppressed tumor growth (Fig. 3 e-g), while no body weight changes were detected among the blank, DMSO, and XCT790-treated groups (Fig. 3 h). To explore the mechanism underlying ESRRA-mediated tumor growth, we performed IHC staining on excised subcutaneous tumors to detect the expression of ESRRA and Ki67 (a well-established marker of cell proliferation). As expected, ESRRA overexpression significantly upregulated Ki67 expression, while XCT790 treatment reduced Ki67 levels (Fig. 3 i-p). This result links ESRRA’s pro-tumorigenic effect to enhanced in vivo cell proliferation—consistent with our earlier in vitro observations on HCC cell proliferation. Moreover, metastasis assays demonstrated that ESRRA overexpression enhanced lung metastasis, as reflected by increased number and size of metastatic nodules. In contrast, XCT790 treatment significantly attenuated metastatic dissemination compared to the DMSO control group (Fig. q-t). In summary, our in vivo data demonstrate that ESRRA functions as a critical regulator of HCC progression by promoting tumor growth and metastasis, while its inhibition effectively suppresses these malignant phenotypes. These findings highlight ESRRA as a potential therapeutic target for HCC intervention. ESRRA activates PHF5A/PI3K/AKT pathway in HCC cells To unravel the molecular mechanism by which ESRRA drives HCC progression, we performed transcriptomic analysis using RNA-Seq. Volcano plots revealed differentially expressed genes (DEGs) between control and ESRRA-knockdown groups (Supplementary Fig. 2a). To identify potential transcriptional targets of ESRRA, we integrated the RNA-Seq data with three independent transcription factor (TF) prediction databases: Gene Transcription Regulation Database (GTRD), hTFtarget, and Cistrome DB (Fig. 4 a, Supplementary Fig. 2b). Next, we used RT-qPCR to validate the overlapping candidate genes from the RNA-seq and database analyses. Among these candidates, PHF5A showed the most robust and consistent regulation across four HCC cell lines: ESRRA knockdown significantly reduced PHF5A mRNA expression, while ESRRA overexpression markedly increased it (Fig. 4 b-c, Supplementary Fig. 2c–f). To confirm this regulation at the protein level, we performed western blotting and IHC staining. Both assays confirmed that PHF5A protein levels closely mirrored its mRNA expression patterns, further supporting ESRRA-dependent regulation of PHF5A (Fig. 4 d, Supplementary Fig. 2g-m). To assess the clinical relevance of PHF5A in HCC, we analyzed public datasets. Data from the HPA and TGCA databases revealed that PHF5A expression was significantly higher in human HCC tissues compared to normal liver tissues (Fig. 4 e–f). Critically, HCC patients with high PHF5A expression had significantly shorter 5-year overall survival (Fig. 4 g), indicating that PHF5A not only correlates with HCC development but also serves as a potential prognostic marker. The binding sites of ESRRA on PHF5A promoter were predicted by JASPAR database ( http://jaspar.genereg.net/ ) (Supplementary Fig. 2n). Notably, the sequence of binding site with the highest score is highly conserved across different host species (Supplementary Fig. 2o), suggesting evolutionary conservation of this regulatory interaction. To validate direct binding, we performed luciferase reporter assays. Cells co-transfecting HCC cells with a wild-type (WT) PHF5A promoter reporter plasmid (PHF5A-WT) and an ESRRA-overexpressing plasmid (pcDNA-ESRRA) resulted in significantly higher luciferase activity compared to co-transfection with PHF5A-WT and empty vector (Fig. 4 h-i). To confirm specificity, we generated PHF5A promoter reporter plasmids with mutations at the three predicted ESRRA binding sites. ESRRA overexpression increased luciferase activity only in cells transfected with PHF5A-WT, not in those with mutations at either site 1 or site 2 (Fig. 4 j–k). These results confirm that ESRRA directly binds to the PHF5A promoter to activate its transcription. To explore the downstream pathway of ESRRA, we performed KEGG analysis. The KEGG pathway analysis of differentially expressed genes downstream of ESRRA identified PI3K/AKT signaling as the most significantly enriched pathways (Fig. 4 i). The result of KEGG analysis was validated by western blotting. Western blotting validated that ESRRA knockdown suppressed PI3K/AKT phosphorylation, while its overexpression activated this axis (Fig. 4 m, Supplementary Fig. 2p-q). Finally, we tested whether PHF5A mediates ESRRA-induced PI3K/AKT activation. PHF5A knockdown significantly suppressed the increased PI3K/AKT phosphorylation triggered by ESRRA overexpression; conversely, PHF5A overexpression rescued the reduced PI3K/AKT phosphorylation caused by ESRRA knockdown (Fig. 4 n, Supplementary Fig. 2r-s). These results demonstrate that PHF5A acts as a critical downstream mediator linking ESRRA to PI3K/AKT activation (Fig. 4 n, Supplementary Fig. 2r-s). Our findings establish ESRRA as a key transcriptional activator of PHF5A, which drives PI3K/AKT signaling activation. ESRRA promotes HCC through PHF5A dependent pathway To determine whether PHF5A mediates the effect of ESRRA on HCC cells, we performed rescue experiments: we overexpressed PHF5A in ESRRA-knockdown HCC cells and silenced PHF5A in ESRRA-overexpressing (OE) HCC cells, then assessed changes in cell proliferation, cell cycle progression, and migration. Strikingly, the overexpression of PHF5A partially reversed the diminished proliferation capacity and the arrested G1 phrases in ESRRA-knocked down cells (Fig. 5 a-d, Supplementary Fig. 3a-b). The silence of PHF5A reversed the increased proliferation of HCC cells induced by ESRRA overexpression and facilitated G1/S transition (Fig. 5 e-h, Supplementary Fig. 3c-d). The overexpression of PHF5A reversed the impaired migration and reduced EMT markers caused by ESRRA knockdown. On the other hand, PHF5A knockdown suppressed the enhanced migration and elevated EMT markers induced by ESRRA overexpression (Fig. 5 i-o, Supplementary Fig. 3e-h). Taken together, these rescue experiments provide direct evidence that ESRRA promotes HCC malignant behaviors—including proliferation, G1/S transition, migration, and EMT—through a PHF5A-dependent pathway. ESRRA promotes HCC development via PHF5A/PI3K/AKT pathway To confirm that the PI3K/AKT pathway is functionally indispensable for ESRRA-mediated promotion of HCC cell proliferation and migration, we performed pharmacologic intervention experiments using the specific PI3K agonist 740 Y-P and the selective PI3K inhibitor LY294002. The proliferation suppression and G1 arrest induced by ESRRA knockdown were effectively rescued by 740Y-P (Fig. 6 a-b). Conversely, the pro-proliferative effect of ESRRA overexpression was abolished by LY294002, as shown in Fig. 6 c-d. Similarly, 740Y-P reversed the migration suppression caused by ESRRA knockdown (Fig. 6 e-j), whereas LY294002 mitigated the enhanced migration due to ESRRA overexpression (Fig. 6 k-p). These results strongly suggest that the PI3K/AKT pathway plays a crucial role in the ability of ESRRA to promote HCC cell proliferation and migration, further supporting the hypothesis that ESRRA promotes HCC development via the PHF5A/PI3K/AKT pathway. Statin inhibits HCC via ESRRA suppression Given that previous studies have demonstrated that cholesterol binds to the ligand-binding domain (LBD) of ESRRA to activate its transcriptional activity, and that statins can reverse this cholesterol-mediated activation[ 23 ], we next investigated whether statins inhibit HCC by suppressing ESRRA function. Among clinically used statins, rosuvastatin was selected for subsequent experiments due to its relatively low liver toxicity[ 24 ]. Firstly, we confirmed that rosuvastatin effectively decreased cholesterol levels in HCC cells (Supplementary Fig. 4a). A time-course transcriptome was performed using dataset GSE24188.We found that in liver cells, the trend of ESRRA mRNA level belonged to cluster 1, which gradually decreased with prolonged rosuvastatin intervention (Supplementary Fig. 4b). RT-qPCR further confirmed that in HCC cells, the mRNA levels of ESRRA declined with extended rosuvastatin treatment, and this effect could be reversed by cholesterol supplementation (Supplementary Fig. 4c-d). Dual-luciferase reporter assays indicated that rosuvastatin treatment markedly downregulated ESRRA transcriptional activity (Fig. 7 a-b). Since statins inhibit cholesterol biosynthesis by targeting the endogenous mevalonate pathway[ 25 ], we performed a rescue experiment by adding exogenous mevalonate to rosuvastatin-treated HCC cells. As expected, mevalonate supplementation reversed the rosuvastatin-induced reduction in ESRRA mRNA levels and transcriptional activity (Fig. 7 a-b). Consistent with these findings, rosuvastatin also downregulated the mRNA level of PHF5A , and this effect was also reversed by mevalonate (Fig. 7 c-d), confirming that statins suppress ESRRA activity via mevalonate pathway inhibition. To confirm that ESRRA mediates the anti-tumor effects of rosuvastatin, we assessed cell proliferation and migration in rosuvastatin-treated cells with or without ESRRA overexpression. ESRRA overexpression completely abolished the anti-proliferative and anti-migratory effects of rosuvastatin (Fig. 7 e-j, Supplementary Fig. 4e-g). Furthermore, mevalonate treatment, which replenished intracellular cholesterol, reversed the anti-proliferation and anti-migratory effects of rosuvastatin in HCC cells. However, this rescue was absent when ESRRA expression was downregulated by XCT790 (an ESRRA-specific antagonist), indicating that the anti-HCC effect of rosuvastatin relied on ESRRA suppression (Fig. 7 k-p, Supplementary Fig. 4h-j). The results above further solidify ESRRA as the key mediator of rosuvastatin’s anti-HCC activity. Discussion In the present research, we discover that ESRRA was upregulated in HCC and promotes HCC cell proliferation and migration both in vitro and in vivo . Mechanistically, ESRRA upregulated PHF5A by directly binding to the promoter, which in turn activated PI3K/AKT pathway. Furthermore, we found that rosuvastatin effectively suppresses the expression and transcriptional activity of ESRRA, which contributes to its anti-tumor effects in HCC cells. Our findings not only identify the ESRRA-PHF5A axis as a potential therapeutic target for HCC but also uncover a novel anti-tumor mechanism of statins in HCC progression, providing a molecular rationale for the clinical application of statins in HCC treatment. The role of ESRRA varies across tumor types, exhibiting both oncogenic and tumor-suppressive functions depending on the cellular context. While it typically promotes tumorigenesis in prostate, ovarian, and esophageal cancers, it paradoxically suppresses tumor growth in triple-negative breast cancer, underscoring the importance of context-dependent regulation[ 10 – 12 , 26 ]. In the present study, we demonstrate that ESRRA functioned as a promoter in HCC proliferation and migration. Intriguingly, this pro-tumorigenic role appears to contradict a previous report showing that ESRRA -null mice exhibit increased susceptibility to liver carcinogenesis. ESRRA deficiency in hepatocytes induces necrosis-driven compensatory proliferation, while its loss in Kupffer cells hyperactivates NF-κB signaling, exacerbating inflammation and tumor initiation[ 27 ]. Together, these seemingly contradictory observations highlight a stage-dependent dual role of ESRRA in HCC pathogenesis. Specifically, during the pre-malignant stages of liver diseases, ESRRA may function as a tumor-suppressive factor by limiting hepatocyte necrosis and suppressing Kupffer cell-mediated inflammation, thereby reducing tumor initiation risk[ 27 ]. In established HCC, however, our data indicate that ESRRA switches to an oncogenic role, driving malignant hepatocyte proliferation and migration. This dual regulatory mechanism highlights the complex, context-specific functions of ESRRA in liver cancer development and progression. Functioning as a transcription factor, ESRRA exerts its effects by regulating downstream target genes. Our RNA-sequencing analysis identified PHF5A —a critical component of the SF3b spliceosomal complex—as a direct transcriptional target of ESRRA in HCC. PHF5A governs alternative splicing, a process frequently dysregulated in cancer[ 28 – 31 ]. Notably, PHF5A is overexpressed in multiple cancers and promotes proliferation and migration[ 32 – 35 ]. However, its role in HCC - particularly its regulatory mechanisms and proliferative effects, remained incompletely understood[ 36 ]. Our study confirmed that ESRRA directly promotes PHF5A transcription in HCC cells, and further demonstrated that PHF5A mediates ESRRA-driven HCC cell proliferation and migration. This discovery thereby uncovers a novel regulatory mechanism responsible for PHF5A upregulation in HCC. While no PHF5A-specific inhibitors currently exist, XCT790 —a well-characterized ESRRA antagonist—has demonstrated efficacy in preclinical models, offering a translational avenue for targeting this axis. Collectively, our study elucidates the upstream regulatory mechanism governing PHF5A upregulation in HCC, which may offer a promising therapeutic strategy for HCC patients with elevated PHF5A expression. The PI3K/Akt pathway is a key signaling pathway driving survival of cancer, including HCC. Its activation promotes HCC cell cycle progression[ 37 ], inhibits cell apoptosis[ 38 ], facilitates angiogenesis and EMT [ 39 – 41 ] and induces drug resistance[ 42 , 43 ]. Our data provide evidence that ESRRA promotes cell proliferation and migration in HCC through activating PI3K/AKT pathway. The present study uncovers ESRRA as a novel upstream regulator that activates this cascade, and highlight ESRRA's significance as a potential new therapeutic target for the aberrant activation of the PI3K/AKT pathway in HCC. Furthermore, activation of the PI3K/AKT pathway is a key factor in the resistance to first - line chemotherapeutic agents such as sorafenib and Lenvatinib, which is a major challenge in HCC treatment[ 43 , 44 ]. Therefore, combining ESRRA inhibitors may relieve sorafenib resistance and enhance the therapeutic efficacy of HCC treatment. Metabolic reprogramming is a hallmark of cancer[ 45 ]. Clinical evidence has demonstrated that metabolism-modulating drugs, including metformin and SGLT2 inhibitors, significantly improve the prognosis of HCC patients, highlighting the pivotal role of metabolic regulation in HCC treatment[ 46 , 47 ]. Alongside glucose metabolism, lipid metabolism has emerged as a critical regulator [ 48 ]. Cholesterol supplies lipids for cell membrane fluidity, signaling transduction, and ECM adhesion, all of which contribute to tumor proliferation and migration in HCC[ 49 – 52 ], while conflicting evidence exists that elevated serum cholesterol enhances natural killer cell-mediated anti-tumor activity and reduces liver tumor growth in mice[ 53 ]. This discrepancy highlights that intracellular cholesterol, rather than extracellular cholesterol, functions as a promoter of HCC progression. Statins, which lower intracellular cholesterol by inhibiting de novo synthesis, thus represent a plausible therapeutic strategy. Clinical studies have confirmed that statins not only reduce HCC incidence risk but also improve HCC patients’ prognosis[ 54 – 56 ]. Furthermore, unlike glucose-lowering drugs-which exert anti-HCC effects primarily in patients with diabetes mellitus, statins can elicit such effects even in individuals without metabolic disorders. Consistent with previous studies, we found that rosuvastatin not only effectively reduced intracellular cholesterol levels in HCC cells but also suppressed their proliferation and migration. Nevertheless, the exact molecular mechanisms of statins' anti - HCC effects remain to be fully elucidated. Previous studies have shown that cholesterol can upregulate ESRRA expression[ 14 , 57 ], or act as a ligand for ESRRA to enhance its transcriptional activity[ 23 ], implying a potential association among cholesterol, statins and ESRRA in HCC. In this study, we found that rosuvastatin can simultaneously suppress both ESRRA expression and its transcriptional activity. This dual-inhibitory effect can be reversed by intracellular cholesterol repletion. Moreover, the anti-HCC effects of rosuvastatin were ESRRA-dependent and could be also reversed by cholesterol replenishment. These results demonstrated that rosuvastatin suppresses HCC by targeting cholesterol - dependent ESRRA activation. To our knowledge, this is the first study to provide mechanistic evidence linking ESRRA inhibition to the anti-HCC effects of statins. Clinically, while several trials have reported that statins have a favorable safety profile and are associated with reduced HCC recurrence and mortality[ 58 – 60 ], another study failed to observe significant therapeutic benefits[ 61 ]. Therefore, additional clinical research is needed to explore whether statin efficacy in HCC correlates with ESRRA expression levels in patients’ tumor tissues. Although we have established the activation of the ESRRA -PHF5A-PI3K/AKT axis, the underlying mechanism by which PHF5A triggers the PI3K/AKT pathway in HCC remains elusive. Previous studies have indicated that PHF5A may regulate downstream genes through alternative splicing[ 62 , 63 ], which might potentially activate this signaling pathway. Therefore, future studies are warranted to explore the detailed interactions between PHF5A and the PI3K/AKT pathway. In addition, while statins have been shown to indirectly suppress ESRRA in the present study, the presence of a ligand - binding domain in ESRRA implies the feasibility of directly targeting it with small - molecule compounds[ 3 ]. Future work could employ molecular simulations and docking to screen compounds that modulate ESRRA activity. In summary, our study has significantly advanced the understanding of the role of ESRRA in HCC, elucidating a novel pathway involving ESRRA, PHF5A and the PI3K/AKT signaling cascade. Specifically, we demonstrated that ESRRA was upregulated in HCC, contributing to its progression via the PHF5A/PI3K/AKT signaling pathway. Moreover, we have established that ESRRA serves as a crucial mediator in the anti - tumor effects of rosuvastatin. As a nuclear receptor, ESRRA can be ligand activated and, therefore, holds great promise as a potential drug target in the future. Declarations Fundings This study was supported by 5010 Cultivation Program of Clinical Research of Sun Yat-Sen University (Grant number: 04A005001000015) and horizontal foundation of Sun Yat-Sen University (Grant number: 06A001001000434). Competing interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions Jiao Gong, Yuankai Wu, Yusheng Jie, and Yutian Chong designed this study and revised the manuscript. Zhiping Wan and Shuying Huang analyzed and interpreted the data. Xiang Cai carried out experiments and wrote the manuscript. 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Mol Cell 74(6):1250–1263 .e6 Supplementary Files SupplementaryMaterials251014.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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-7867831","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":535693401,"identity":"0a840a62-5507-4f78-9b55-79dd2c532127","order_by":0,"name":"Xiang Cai","email":"","orcid":"","institution":"Third Affiliated Hospital of Sun Yat-Sen University","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Cai","suffix":""},{"id":535693402,"identity":"55241856-a883-4234-8446-68bb20d15dab","order_by":1,"name":"Zhiping Wan","email":"","orcid":"","institution":"Third Affiliated Hospital of Sun Yat-Sen 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1","display":"","copyAsset":false,"role":"figure","size":11364223,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEstrogen related receptor alpha (ESRRA) facilitates HCC cell proliferation in vitro.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) ESRRA mRNA expression levels in normal liver tissues and HCC tissues from The Cancer Genome Atlas (TCGA) database.\u003c/p\u003e\n\u003cp\u003e(b) Western blot analysis of ESRRA protein expression in the non-malignant hepatic cell line THLE-2 and indicated hepatocellular carcinoma (HCC) cell lines. GAPDH served as the loading control. Representative blot shown from three independent experiments.\u003c/p\u003e\n\u003cp\u003e(c, d) Colony formation assays following ESRRA knockdown (si1, si2) in HepG2 (c) and MHCC-97H.\u003c/p\u003e\n\u003cp\u003e(d) cells compared to negative control (NC).\u003c/p\u003e\n\u003cp\u003e(e, f) EdU proliferation assays in ESRRA-knockdown HepG2 (e) and MHCC-97H (f) cells vs. NC. Images captured at 20× magnification; scale bar = 100 µm.\u003c/p\u003e\n\u003cp\u003e(g, h) Cell proliferation assessed by CCK-8 assays in HepG2 (g) and MHCC-97H (h) cells after ESRRA knockdown. (*p \u0026lt; 0.05, si1 vs. NC; #p \u0026lt; 0.05, si2 vs. NC).\u003c/p\u003e\n\u003cp\u003e(i, j) Cell cycle distribution analysis and quantification in HepG2 (i) and MHCC-97H (j) cells after ESRRA knockdown. (\u003csup\u003e%\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, %G1 phase; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, %S phase; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, %G2/M phase vs. NC).\u003c/p\u003e\n\u003cp\u003e(k, l) Colony formation assays following ESRRA overexpression (ESRRA-OE) in Huh7 (k) and SNU-449 (l) cells compared to empty vector control (EV).\u003c/p\u003e\n\u003cp\u003e(m, n) EdU proliferation assays in ESRRA-overexpressing Huh7 (m) and SNU-449 (n) cells vs. EV. Images captured at 20× magnification; scale bar = 100 µm.\u003c/p\u003e\n\u003cp\u003e(o, p) CCK-8 assays assessing proliferation of Huh7 (o) and SNU-449 (p) cells after ESRRA overexpression. \u0026nbsp;*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, EV group vs. ESRRA group.\u003c/p\u003e\n\u003cp\u003e(q, s) Cell cycle analyses and statistical results of Huh7 (q) and SNU-449 (s) cells after ESRRA overexpression. (\u003csup\u003e%\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, %G1 phase; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, %S phase; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, %G2/M phase vs. EV).\u003c/p\u003e\n\u003cp\u003eData represent mean ± SD from biological replicates. (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Abbreviations: ESRRA, Estrogen Related Receptor Alpha; EV, Empty Vector; HCC, Hepatocellular Carcinoma; NC, Negative Control; si1, ESRRA siRNA (1); si2, ESRRA siRNA (2); TCGA, The Cancer Genome Atlas.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7867831/v1/433e42f572560433c416f005.png"},{"id":95298116,"identity":"aa2a379a-97e1-42f4-8612-c65de917795b","added_by":"auto","created_at":"2025-11-06 12:43:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":15214928,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eESRRA accelerates HCC cell migration \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(a,b) Representative images from wound healing assays assessing migration in HepG2 (a) and MHCC-97H (b) cells following ESRRA knockdown (si1, si2) compared to negative control (NC). Images captured at 10× magnification; scale bar = 200 µm.\u003c/p\u003e\n\u003cp\u003e(c) Quantification of relative wound closure in HepG2 and MHCC-97H cells after ESRRA knockdown (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e(d) Representative images and quantification of Transwell migration assays in ESRRA-knockdown HepG2 and MHCC-97H cells vs. NC. Images captured at 20× magnification; scale bar = 100 µm. (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e(e) Western blot analysis of EMT marker protein expression (N-cadherin, E-cadherin, Vimentin) and quantification in ESRRA-knockdown HepG2 and MHCC-97H cells. GAPDH served as loading control. Representative blot shown from three independent experiments. (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. NC).\u003c/p\u003e\n\u003cp\u003e(f) Representative images and quantification of wound healing assays in Huh7 and SNU-449 cells after ESRRA overexpression (ESRRA-OE) vs. empty vector control (EV). Images captured at 10× magnification; scale bar = 200 µm. (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e(g) Representative images and quantification of transwell migration assays in ESRRA-overexpressing Huh7 and SNU-449 cells vs. EV. Images captured at 20× magnification; scale bar = 100 µm. (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e(h) Western blot analysis of EMT marker protein expression and quantification in ESRRA-overexpressing Huh7 and SNU-449 cells. GAPDH served as loading control. (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. EV).\u003c/p\u003e\n\u003cp\u003eData represent mean ± SD from three independent experiments. Abbreviations:\u0026nbsp; NC, negative control; si1, ESRRA siRNA (1); si2, ESRRA siRNA (2); EV, empty vector, ESRRA, ESRRA overexpression; ERRα, ESRRA; N-cad, N-cadherin; E-cad, E-cadherin; VIM, Vimentin.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7867831/v1/ee5cbc1da5cd61740c71c7ab.png"},{"id":95314333,"identity":"8a308d12-8468-4541-b010-cc21c196e818","added_by":"auto","created_at":"2025-11-06 15:52:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":20391782,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eESRRA accelerates HCC cell migration \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a-c) Subcutaneous xenografts volume and weight over time in nude mice injected with Huh7 cells from blank group, empty vector group, and ESRRA overexpression group (n=5). *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, EV group vs. ESRRA group.\u003c/p\u003e\n\u003cp\u003e(d) Body weights of tumor-bearing mice injected with MHCC-97H cells.\u003c/p\u003e\n\u003cp\u003e(e-g) Subcutaneous xenografts volume and weight over time in nude mice injected with MHCC-97H cells from blank group, DMSO group, and XCT790 treatment group (n=5). *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, DMSO group vs. XCT790 group.\u003c/p\u003e\n\u003cp\u003e(h) Body weights of tumor-bearing mice injection with MHCC-97H cells.\u003c/p\u003e\n\u003cp\u003e(i-p) Representative immunohistochemical staining and quantitative analysis of ESRRA and Ki-67 expression in xenograft tissues (20× magnification; scale bar = 100 μm).\u003c/p\u003e\n\u003cp\u003e(q-r) Representative Haematoxylin/eosin-stained images of lung sections showing metastatic foci (5× magnification; scale bar = 200 μm).\u003c/p\u003e\n\u003cp\u003e(s-t) Quantification of lung metastatic nodules (n=5).\u003c/p\u003e\n\u003cp\u003eData represent mean ± SD; *p\u0026lt;0.05. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05. Abbreviations: DMSO, dimethyl sulfoxide; EV, empty vector; ERRα, ESRRA overexpression.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7867831/v1/f4c1afdb4edc4c818d1db3df.png"},{"id":95314561,"identity":"04811542-ff1b-49ea-863c-12f32a1cd20d","added_by":"auto","created_at":"2025-11-06 15:53:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5659679,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eESRRA activates PHF5A/PI3K/AKT pathway in HCC cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) ESRRA target gene prediction using three public databases.\u003c/p\u003e\n\u003cp\u003e(b-c) RT-qPCR analysis of PHF5A mRNA levels following ESRRA knockdown (KD) or ESRRA overexpression (OE) in HCC cells. GAPDH served as the endogenous control. (*\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e(d) Western blot analysis of PHF5A protein levels after ESRRA KD or OE. GAPDH was the loading control. Data represent three independent experiments.\u003c/p\u003e\n\u003cp\u003e(e) ESRRA expression in normal vs. HCC tissues (TCGA database).\u003c/p\u003e\n\u003cp\u003e(f) The expression level of ESRRA in normal and tumor tissues in TGCA database. (*\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, vs. Normal group).\u003c/p\u003e\n\u003cp\u003e(g) Overall survival analysis of HCC patients stratified by ESRRA expression.\u003c/p\u003e\n\u003cp\u003e(h-i) Dual-luciferase reporter assay of PHF5A promoter activity co-transfected with EV or ESRRA-OE plasmid (n=3). (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e(j-k) Dual-luciferase reporter assay of ESRRA co-transfected with wild-type (WT) PHF5A promoter or binding site mutants (MUT1-3) (n=3). (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, NS=Not Significant).\u003c/p\u003e\n\u003cp\u003e(l) Bubble chart of top 10 enriched KEGG pathways for downregulated DEGs\u003c/p\u003e\n\u003cp\u003e(m) Western blot analysis of PI3K and AKT (total/phosphorylated) levels in HepG2 (ESRRA-KD) and Huh7 (ESRRA-OE) cells. GAPDH was the loading control. Data represent three independent experiments.\u003c/p\u003e\n\u003cp\u003e(n) Western blot analysis of PI3K/AKT signaling in HepG2 cells (ESRRA KD ± PHF5A rescue) and Huh7 cells (ESRRA-OE ± PHF5A KD). GAPDH was the loading control. Data represent three independent experiments. (HepG2 cells: 1: NC group, 2: siESRRA group, 3: siESRRA+ EV group, 4: siESRRA + PHF5A-OE group; Huh7 cells: a: EV group, b: ESRRA-OE group, c: ESRRA-OE + siNC group, d: ESRRA-OE + siPHF5A1 group, e: ESRRA-OE + siPHF5A2 group)\u003c/p\u003e\n\u003cp\u003eFor all panels, data are presented as mean values ± SD. Abbreviations: DEG, differentially expressed gene; EV, empty vector; HPA, Human Protein Atlas; IHC, immunohistochemistry; KD, knockdown; KEGG, Kyoto Encyclopedia of Genes and Genomes; MUT, mutant; NC, negative control; OE, ESRRA overexpression; RT-qPCR, quantitative real-time PCR; TCGA, The Cancer Genome Atlas; WT, wild-type.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7867831/v1/c260eacee6d8f233051c7842.png"},{"id":95298123,"identity":"ee555d99-5a42-40cc-8500-8ae5b117963f","added_by":"auto","created_at":"2025-11-06 12:43:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":14355088,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eESRRA promotes HCC through PHF5A dependent pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a-b) HepG2 proliferation assessed by CCK-8 (a) and colony formation (b) assays in the following groups: Group 1: NC; Group 2: siESRRA; Group 3: siESRRA + EV; Group 4: siESRRA + PHF5A-OE. (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 NC vs. siESRRA, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 siESRRA+PHF5A-OE vs. siESRRA).\u003c/p\u003e\n\u003cp\u003e(c) Representative images (20×; scale bar: 100 μm) of EdU proliferation assay in HepG2 (groups as a-b).. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(d) Cell cycle distribution in HepG2 (groups as a-b).\u003csup\u003e %\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (%G1), \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (%S), \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (%G2), compared with group 1;\u003csup\u003e +\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (%G1), \u003csup\u003eΔ\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (%S), \u003csup\u003e▲\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (%G2), compared with group 2.\u003c/p\u003e\n\u003cp\u003e(e-f) Colony formation (e) and CCK-8 (f) assays in Huh7 cells in the following groups: Group a: EV; Group b: ESRRA-OE; Group c: ESRRA-OE + siNC; Group d: ESRRA-OE + siPHF5A (1); Group e: ESRRA-OE + siPHF5A (2). (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 EV vs. ESRRA-OE; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 ESRRA+siPHF5A (1) vs. ESRRA-OE; \u003csup\u003e%\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 ESRRA+siPHF5A (2) vs. ESRRA-OE).\u003c/p\u003e\n\u003cp\u003e(g) Representative images (20×; scale bar: 100 μm) of EdU proliferation in Huh7 (groups as e-f).\u003c/p\u003e\n\u003cp\u003e(h) Cell cycle analysis in Huh7 in the following groups: Group a: EV; Group b: ESRRA-OE; Group c: ESRRA-OE + siNC; Group d: ESRRA-OE + siPHF5A (1); Group e: ESRRA-OE + siPHF5A (2). \u003csup\u003e%\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (%G1), \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (%S), \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (%G2), compared with group a.\u003csup\u003e %\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (%G1), \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (%S), \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (%G2), \u003csup\u003e+\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (%G1), \u003csup\u003eΔ\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (%S), \u003csup\u003e▲\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (%G2), compared with group b.\u003c/p\u003e\n\u003cp\u003e(i-j) Representative images (10×; scale bar: 200 μm) of wound healing assays of HepG2 (i) and Huh7 (j) cells in each group.\u003c/p\u003e\n\u003cp\u003e(k-l) Representative images (10×; scale bar: 200 μm) of transwell assays in HepG2 (k) and Huh7 (l) (groups as h).\u003c/p\u003e\n\u003cp\u003e(m-o) EMT marker expression in HepG2 and Huh7 cells (groups as above). GAPDH was the loading control. Data represent three independent experiments. (NS, Not Significant. For HepG2: \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. NC, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, vs. siESRRA+PHF5A-OE; for Huh7: \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. EV, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, vs. ESRRA-OE+siPHF5A (1), \u003csup\u003e%\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. ESRRA-OE+siPHF5A (2).\u003c/p\u003e\n\u003cp\u003eFor all panels, data are presented as mean values ± SD. Abbreviations: NC, negative control; siESRRA, ESRRA siRNA; EV, empty vector; PHF, PHF5A overexpression; ESRRA, ESRRA overexpression; si1, PHF5A siRNA (1); si2, PHF5A siRNA (2).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7867831/v1/96dec93fc1f54a9ce0be843a.png"},{"id":95298120,"identity":"a9ec5eef-3e59-444a-840d-75060cfc5fee","added_by":"auto","created_at":"2025-11-06 12:43:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":13832238,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eESRRA promotes HCC development via PHF5A/PI3K/AKT pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) CCK-8 assay showing HepG2 proliferation after ESRRA knockdown ± PI3K agonist 740Y-P. (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 NC vs. siESRRA, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 siESRRA+740 Y-P vs. siESRRA).\u003c/p\u003e\n\u003cp\u003e(b) Representative images (20×; scale bar: 100 μm) of EdU proliferation assays in HepG2 (siESRRA ± 740Y-P) with quantification. (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. NC, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. siESRRA+740 Y-P).\u003c/p\u003e\n\u003cp\u003e(c) CCK-8 assay showing Huh7 proliferation (ESRRA-OE ± LY294002). \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ESRRA vs. EV, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ESRRA group vs. ESRRA + LY group. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(d) Representative images (20×; scale bar: 100 μm) of EdU proliferation assays in Huh7 (ESRRA-OE ± LY294002) with quantification. (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. EV, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. ESRRA + LY). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(e-f) Representative images (20×; scale bar: 100 μm) of transwell analyses in HepG2 (siESRRA ± 740Y-P) cells with quantification. (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, vs. NC group, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. siESRRA+740 Y-P).\u003c/p\u003e\n\u003cp\u003e(g-h) Representative images (10×; scale bar: 200 μm) of wound healing assays of HepG2 cells (siESRRA ± 740Y - P) with quantification. (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, vs. NC group, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. siESRRA+740 Y-P).\u003c/p\u003e\n\u003cp\u003e(i-j) Western blot analysis of EMT markers (E-cadherin, N-cadherin, Vimentin) in HepG2 cells (1: NC; 2: siESRRA; 3: siESRRA+DMSO; 4: siESRRA+740Y-P). \u0026nbsp;GAPDH was the loading control. Data represent three independent experiments. \u0026nbsp;(\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, vs. NC, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. siESRRA+740 Y-P).\u003c/p\u003e\n\u003cp\u003e(k-l) Representative images (20×; scale bar: 100 μm) of transwell analyses in Huh7 (ESRRA-OE ± LY294002) with quantification. (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. EV, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. ESRRA + LY).\u003c/p\u003e\n\u003cp\u003e(m-n) Representative images (10×; scale bar: 200 μm) of wound healing assays in Huh7 cells (a: EV; b: ESRRA-OE; c: ESRRA-OE+DMSO; d: ESRRA-OE+LY294002) with quantification. (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. EV, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. ESRRA + LY).\u003c/p\u003e\n\u003cp\u003e(o-p) Western blot analysis of EMT markers (E-cadherin, N-cadherin, Vimentin) in Huh7 cells (a: EV; b: ESRRA-OE; c: ESRRA-OE+DMSO; d: ESRRA-OE+LY294002). GAPDH was the loading control. Data represent three independent experiments.\u0026nbsp; (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. EV, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. ESRRA + LY).\u003c/p\u003e\n\u003cp\u003eFor all panels, data are presented as mean values ± SD. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05. Abbreviations: NC, negative control; si, ESRRA siRNA; EV, empty vector; LY, LY294002; NS, not significant.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7867831/v1/459551ab2507f5483c82279e.png"},{"id":95314834,"identity":"26745d30-2415-40fc-b67b-e363c0b79704","added_by":"auto","created_at":"2025-11-06 15:53:22","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":17894962,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStatin inhibits HCC via ESRRA suppression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a-b) The ERRE - luciferase activity was determined using a dual - luciferase reporter assay system in HepG2 and Huh7 cells. Cells were transfected with ESRRA plasmid or empty vector, followed by treatment with rosuvastatin (Rosu) ± mevalonate (MEVA). (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, NS = Not Significant).\u003c/p\u003e\n\u003cp\u003e(c-d) RT-qPCR analysis of PHF5A mRNA levels in HepG2 and Huh7 cells treated with Rosu ± MEVA. GAPDH: internal control. \u0026nbsp;(\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, NS = Not Significant).\u003c/p\u003e\n\u003cp\u003e(e-f) CCK-8 proliferation assays in HepG2 and Huh7 cells (transfected with ESRRA or EV) treated with Rosu. (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 DMSO vs. Rosu, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 Rosu + ESRRA vs. Rosu).\u003c/p\u003e\n\u003cp\u003e(g) Representative images (20×; scale bar: 100 μm) of EdU proliferation assays in HepG2 and Huh7 cells (transfected with ESRRA or EV) treated with Rosu.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(h-i) Representative images (10×; scale bar: 200 μm) of wound healing analyses in HepG2 and Huh7 cells (Rosu ± ESRRA). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(j) Representative images (20×; scale bar: 100 μm) of transwell assays in HepG2 and Huh7 cells (Rosu ± ESRRA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(k-l) CCK - 8 assays of HepG2 and Huh7 cells in the following four groups: DMSO, Rosu, Rosu + MEVA, Rosu + MEVA + XCT790. (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, DMSO vs. Rosu, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, Rosu + MEVA vs. Rosu, \u003csup\u003e%\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, Rosu + MEVA vs. Rosu + MEVA + XCT).\u003c/p\u003e\n\u003cp\u003e(m) Representative images (20×; scale bar: 100 μm) of EdU assays of cell proliferation in HepG2 and Huh7 cells (groups as k-l).\u003c/p\u003e\n\u003cp\u003e(n) Representative images (20×; scale bar: 100 μm) of transwell assays in HepG2 and Huh7 cells (groups as k-l).\u003c/p\u003e\n\u003cp\u003e(o-p) Representative images (10×; scale bar: 200 μm) of wound healing analyses of HepG2 and Huh7 cells (groups as k-l).\u003c/p\u003e\n\u003cp\u003eData are presented as mean values ± SD. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05. Abbreviations: Rosu, rosuvastatin; MEVA, mevalonate; EV, empty vector; XCT, XCT790 (ESRRA inhibitor); NS, not significant.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7867831/v1/dcc28ea849cd777f3be6f955.png"},{"id":95314640,"identity":"6df226ab-bd77-4ce6-9c16-1e34106d311b","added_by":"auto","created_at":"2025-11-06 15:53:08","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":4028485,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract outlining the proposed mechanism.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eESRRA promotes cell proliferation and migration in HCC cells by transcriptionally upregulating PHF5A and subsequently activates PI3K/AKT pathway. Rosuvastatin downregulates the mRNA expression and suppresses the transcriptional activity of ESRRA by lowering intracellular cholesterol, a endogenous ligand of ESRRA.\u003c/p\u003e","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-7867831/v1/4f73181e32bd5baa3ae9244d.png"},{"id":101207140,"identity":"7813f450-1a7a-4b30-bb49-fc64ec64e634","added_by":"auto","created_at":"2026-01-27 09:57:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":98554686,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7867831/v1/2fb826f3-24fd-477e-9183-1472c9583f48.pdf"},{"id":95314619,"identity":"3f3f29b3-b855-4846-9df9-48d78f070bf1","added_by":"auto","created_at":"2025-11-06 15:53:06","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2285387,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials251014.docx","url":"https://assets-eu.researchsquare.com/files/rs-7867831/v1/9cbe31f7a21969bfee3e3603.docx"}],"financialInterests":"","formattedTitle":"ESRRA-Driven PHF5A Activation Promotes Hepatocellular Carcinoma Progression and is Therapeutically Targeted by Rosuvastatin","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLiver cancer exhibits a concerning global health burden, ranking as the sixth most prevalent malignancy and the third leading cause of cancer-related deaths worldwide, imposing substantial socioeconomic and healthcare challenges[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Among primary liver cancers, hepatocellular carcinoma (HCC) is the most prevalent histological subtype. Therefore, elucidating the molecular mechanisms underlying HCC progression, identifying innovative therapeutic strategies, and optimizing existing treatment modalities are imperative.\u003c/p\u003e\u003cp\u003eThe superfamily of nuclear receptors (NRs), which consists of transcriptional factors, has been extensively investigated regarding their functions in the development and progression of cancer [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Additionally, NRs possess unique ligand-binding sites that can interact with small molecules. This characteristic has rendered them appealing targets for the development of cancer treatments [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The roles of NRs in HCC vary. Some promote HCC progression, such as Nuclear Receptor Subfamily 4 Group A Member 3 (NR4A3) and Nuclear Receptor Subfamily 2 Group E (e.g., HNF4α), while others suppress it, like Nuclear Receptor Subfamily 0, Group B, Member 1 (NR0B1)[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The estrogen receptor (ER)-related receptors (ERRs), including ESRRA, ESRRB, and ESRRG, constitute the NR3B subgroup of NRs and play pivotal roles in regulating cellular metabolism and development[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Among these, ESRRA is predominantly expressed in tissues with high energy demands, while ESRRB is active during early embryonic development, and ESRRG is primarily found in the central nervous system and spinal cord [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Notably, ESRRA directly regulates key metabolic pathways, such as mitochondrial biogenesis, oxidative phosphorylation, and glycolysis, which were required for the proliferation and migration of cancer cells[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Given that cancer cells similarly exhibit heightened energy requirements, it is unsurprising that ESRRA\u0026mdash;more than other ERRs\u0026mdash;has been extensively linked to various cancers, including breast, ovarian, prostate, and esophageal cancers [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, despite its well-established role in other cancers, the role of ESRRA in HCC remains underexplored.\u003c/p\u003e\u003cp\u003eConsequently, the present study aims to elucidate the precise role of ESRRA in the development of HCC and the underlying molecular mechanisms. Moreover, it may help to identify novel and potentially effective targets for the treatment of HCC.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell culture\u003c/h2\u003e\u003cp\u003eThe human normal liver epithelial cell line (THLE2) and hepatocellular carcinoma (HCC) cell lines (HepG2, MHCC-97H, Huh7, and SNU-449) were obtained from the Cell Bank of the Chinese Academy of Sciences. All cell lines were authenticated by short tandem repeat (STR) profiling prior to experimental use. THLE2, HepG2, and MHCC-97H cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM; Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS; Procell, Wuhan, China). SNU-449 cells were maintained in RPMI-1640 medium (Gibco, CA, USA) containing 10% FBS (Procell). All cells were incubated at 37\u0026deg;C in a humidified atmosphere with 5% CO2.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003esiRNA and plasmid construction\u003c/h3\u003e\n\u003cp\u003esiRNAs targeting ESRRA and PHD finger protein 5A (PHF5A), along with a negative control siRNA, were synthesized by Hanyi Biotechnology (Shanghai, China) (sequences provided in Supplementary Table\u0026nbsp;1). For overexpression studies, the pcDNA3.1-ESRRA and pcDNA3.1-PHF5A plasmids, as well as the empty vector control (pcDNA3.1), were obtained from Genepharma (Shanghai, China) (plasmid sequences detailed in Supplementary Tables\u0026nbsp;2 and 3). Transfections were performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer\u0026rsquo;s protocol. Briefly, 50 nM siRNA or 2 \u0026micro;g plasmid DNA was diluted in Opti-MEM, mixed with Lipofectamine 3000 reagent (1:1 ratio, v/v), and incubated for 15 min at room temperature (RT) before being added to the cells. The knockdown efficiency (for siRNA) and overexpression efficiency (for plasmids) were confirmed by Western blot analysis.\u003c/p\u003e\n\u003ch3\u003eProtein Extraction and Western Blot Analysis\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eProtein Extraction and Western Blot Analysis\u003c/div\u003e\u003cp\u003eTotal protein was extracted using RIPA lysis buffer supplemented with protease inhibitors. The lysates were then separated by SDS-PAGE and electrophoretically transferred onto PVDF membranes. To minimize nonspecific binding, the membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature. Subsequently, the membranes were incubated with primary antibodies overnight at 4\u0026deg;C, followed by 1 h incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature. Protein signals were visualized using an enhanced chemiluminescence (ECL) detection system (ZETA LIFE). Detailed information on the antibodies used is provided in Supplementary Table\u0026nbsp;4.\\\u003c/p\u003e\n\u003ch3\u003eColony formation assay\u003c/h3\u003e\n\u003cp\u003eHCC cells were digested by trypsin, counted, and seeded at a low density (typically 500\u0026ndash;1000 cells per well) in 6-well plates containing complete growth medium. The plates were then incubated at 37\u0026deg;C in a humidified atmosphere with 5% CO₂ for two weeks, allowing colony formation. After that, cells were gently washed with phosphate-buffered saline (PBS) for 3 times, fixed with 4% paraformaldehyde for 20 min, and stained with crystal violet for 30 min at room temperature. Excess stain was removed by washing with distilled water, and plates were air-dried. Colonies consisting of more than 50 cells were counted.\u003c/p\u003e\n\u003ch3\u003eCell Counting Kit-8 (CCK-8) Assay\u003c/h3\u003e\n\u003cp\u003eHCC cells were seeded in 96-well plates at a density of 3,000\u0026ndash;5,000 cells/well in 100 \u0026micro;L of complete culture medium and allowed to adhere. At days 0, 1, 2, and 3, 10 \u0026micro;L of CCK-8 solution was added to each well, followed by incubation at 37\u0026deg;C for 2 h. Absorbance was measured at 450 nm using a Tecan SPARK microplate reader (Switzerland).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eEdU proliferation assay\u003c/h2\u003e\u003cp\u003eHCC cells were seeded in 48-well plates (or chamber slides) in complete growth medium. After 48 h of culture, cells were incubated with EdU solution (50 \u0026micro;M, BeyoClick\u0026trade; EdU Kit, Beyotime, Shanghai, China) for 2\u0026ndash;4 h. Subsequently, cells were washed by PBS, and fixed with 4% paraformaldehyde for 20 min. Permeabilization was performed using 0.5% Triton X-100 in PBS for 30 min. The Click-iT\u0026reg; reaction cocktail was prepared according to the manufacturer\u0026rsquo;s protocol. Cells were incubated with the reaction mixture for 30 min (protected from light). Cells were washed and counterstained with Hoechst 33342 (1 \u0026micro;g/mL, 10 min, RT) for nuclear visualization. Fluorescence images were captured using a Zeiss inverted fluorescence microscope at 20\u0026times; magnification.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eWound-healing assay\u003c/h3\u003e\n\u003cp\u003eHCC cells were seeded in 6-well plates at a density of 5 \u0026times; 10⁵ cells/well in complete growth medium until reaching 90\u0026ndash;100% confluence. A sterile 20 \u0026micro;L pipette tip was used to create a straight scratch wound in the cell monolayer. After that, the cells were gently washed with PBS to remove detached cells and incubated with 2% FBS-supplemented medium. Images were captured after 24 h using a Zeiss inverted fluorescence microscope (10\u0026times;). Wound healing magnitude was quantified by measuring the relative wound closure compared with control cells at 24 hours[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eTranswell migration assay\u003c/h3\u003e\n\u003cp\u003eHCC cells were harvested and resuspended in serum-free medium. Polycarbonate membrane inserts (8-\u0026micro;m pore size) were placed in 24-well plates. The lower chamber was filled with 600 \u0026micro;L of chemoattractant medium (DMEM with 10% FBS). 100 \u0026micro;L of cell suspension was added to the upper chamber. Cells were incubated at 37\u0026deg;C for 24 hours. Non-migrated cells on the upper membrane surface were removed. Migrated cells on the lower membrane surface were fixed with 4% paraformaldehyde and stained with crystal violet for 15 min. Migrated cells were counted under a Zeiss inverted fluorescence microscope at 20\u0026times; fields.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eCell cycle analysis\u003c/h2\u003e\u003cp\u003eHCC cells were harvested, washed twice with ice-cold PBS, and fixed in 70% ice-cold ethanol at 4\u0026deg;C overnight. Fixed cells were resuspended in PBS and treated with RNase A at 37\u0026deg;C for 30 min. Afterwards, cells were stained with propidium iodide (Solarbio, Beijing, China) in the dark for 30 min. Finally, the cell cycle distributions of the samples were detected by flow cytometry (Cytek Biosciences, Fremont, CA). The cell cycle distribution was analyzed using the software FlowJo_v10.9.0 (Oregon, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003eAnimal experiments\u003c/em\u003e\u003c/h2\u003e\u003cp\u003e Animal experiments were conducted with strict adherence to ethical and scientific standards. BALB/c nude male mice aged 3 weeks were obtained from GemPharmatech Co., Ltd (Nanjing, China). All procedures involved in the animal experiments were approved by the Committee of Animal Experimental Ethics (IACUC \u0026minus;\u0026thinsp;202505166). The mice were housed in specific pathogen - free (SPF) facilities under well - controlled conditions, including a 12 - h light/dark cycle, a temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, and a humidity of 50\u0026thinsp;\u0026plusmn;\u0026thinsp;10%, with unrestricted access to autoclaved food and water.\u003c/p\u003e\u003cp\u003eTo establish subcutaneous tumor models in nude mice, MHCC-97H and Huh7 cells were seeded in 10-cm culture dishes. The Huh7 cells were infected with either the negative lentivirus or the lentivirus-based ESRRA overexpression vector from Jima Company (Shanghai, China). The sequence of the ESRRA lentivirus is detailed in Supplementary Table\u0026nbsp;5. Subsequently, selection was carried out in a medium containing puromycin (2\u0026micro;g/ml) from Jima Company (Shanghai, China) to obtain stable transfectants. When the cells reached 80\u0026ndash;90% confluency, the MHCC \u0026minus;\u0026thinsp;97H and Huh7 cells were harvested using 0.25% trypsin-EDTA and resuspended. After a 7-day acclimatization period, 5\u0026times;10⁶ HCC cells in a 100\u0026micro;L suspension were subcutaneously injected into the right flank of the BALB/c nude male mice. The mice were monitored daily for tumor growth and overall health status. Ten days after the subcutaneous injection of MHCC-97H cells, the tumor - bearing mice were randomly divided into three groups: the XCT790 group, which received an intraperitoneal injection of 8 mg/kg XCT790 dissolved in dimethyl sulfoxide (DMSO); the vehicle control group, which was given an equivalent volume of DMSO; and the blank control group, which received an equivalent volume of PBS[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Tumor volume was measured every 3\u0026ndash;4 days using calipers and calculated using the formula: tumor volume = (length \u0026times; width\u0026sup2;)/2. For the Huh7 xenografts, the tumors were removed 4 weeks after injection. For the MHCC-97H models, the tumors were harvested 3 weeks after XCT790 treatment. Prior to tumor collection, the mice were euthanized by CO₂ asphyxiation.\u003c/p\u003e\u003cp\u003eFor the lung metastasis model, MHCC-97H cells and Huh7 cells (from both empty vector and ESRRA overexpression groups) were collected and injected via the tail vein into nude mice (2\u0026times;10⁶ cells per mouse). Two weeks later, mice injected with MHCC-97H cells were divided into two groups: XCT790 group (8 mg/kg) and DMSO group (equivalent volume of vehicle), with treatments administered via intraperitoneal injection. Five weeks after tail vein injection, all mice (injected with either MHCC-97H or Huh7 cells) were euthanized by CO₂ asphyxiation, and lung tissues were isolated for subsequent hematoxylin-eosin (HE) staining.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemistry (IHC) analysis\u003c/h2\u003e\u003cp\u003eSubcutaneous tumor tissues were fixed in neutral buffered formalin for 24 h at room temperature, followed by dehydration through a graded ethanol series. Tissues were cleared in xylene, infiltrated with paraffin, and embedded in paraffin blocks. The paraffin-embedded tumor tissues were cut into 4-\u0026micro;m-thick sections, dewaxed with xylene and rehydrated with descending ethanol. After antigen retrieval, the tissue slides were blocked with 5% bovine serum albumin (BSA) at room temperature and then Incubated overnight at 4\u0026deg;C with the primary antibodies anti-ki67, anti-ESRRA, and anti-Plant Homeodomain Finger Protein 5A (PHF5A). The detailed information of antibodies was listed in Supplementary Table\u0026nbsp;6.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eHE staining\u003c/h2\u003e\u003cp\u003eUpon euthanasia, lung tissues from each group of nude mice were fixed in 10% formalin for 48 hours, paraffin-embedded, and sectioned at 4 \u0026micro;m thickness. The sections were mounted on glass slides, dewaxed in xylene, and rehydrated through a graded ethanol series. For histological staining, sections were treated with hematoxylin for 2 minutes at room temperature, rinsed under running water for 5 minutes to restore a blue hue, and counterstained with eosin for 5 minutes. Finally, slides were dehydrated through an ascending ethanol series, cleared in xylene, and prepared for analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eRNA sequencing\u003c/h2\u003e\u003cp\u003eRNA sequencing and data analysis were performed according to our previously published research and was performed by Hangzhou Lianchuan Bio Technologies Co[\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Briefly, total RNA was extracted by TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Each sample in normal control (NC) group and ESRRA siRNA group were resultant mix of nine RNA extraction. The total RNA quantity and purity were analyzed with a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA). RNA fragments are enriched by oligo (dT) magnetic beads (Dynabeads Oligo (dT), Thermo Fisher, USA). Finally, RNA sequencing was performed using Illumina Novaseq\u0026trade; 6000 platform.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eQuantitative real-time PCR\u003c/h2\u003e\u003cp\u003eFor quantitative real - time PCR analysis, total RNA was isolated from the cultured cell lines using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Subsequently, complementary DNA (cDNA) was synthesized with a commercial reverse transcription kit according to the manufacturer\u0026rsquo;s instructions. (Takara, Tokyo, Japan). After cDNA synthesis, quantitative PCR amplification was carried out using a Light Cycler 480II Real-Time PCR System (Roche Diagnostics, Mannheim, Germany). This was achieved by using SYBR Green Master Mix (TAKARA) on the LightCycler 480 instrument (Roche, Basel, Switzerland). The sequences of all PCR primers employed in this experiment are detailed in Supplementary Table\u0026nbsp;7.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eDual luciferase reporter assay\u003c/h2\u003e\u003cp\u003eThe binding sites of ESRRA at the PHF5A promoter were predicted using the JASPAR database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://jaspar.genereg.net/\u003c/span\u003e\u003cspan address=\"https://jaspar.genereg.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Luciferase reporter assays were performed following previously published protocols[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The wild-type or mutant promoters of human \u003cem\u003ePHF5A\u003c/em\u003e were transduced into pGL3-basic vectors (Supplementary table 8\u0026ndash;12). The ESRRA expression plasmid or empty vector, PGC1-α expression plasmid, and wild-type or mutant pGL3-basic vectors were co-transfected into HCC cells. To assess the transcriptional activity of ESRRA, the ESRRA expression plasmid or an empty vector was co-transfected with the ERRE-luc reporter (Yeasen, Shanghai, China) into HCC cells. These HCC cells were pre-treated under three distinct conditions: with rosuvastatin (25 \u0026micro;M, MCE, New Jersey, USA), with a combination of rosuvastatin and mevalonate (3 mM, Sigma-Aldrich, Saint Louis, USA), or with an equal volume of DMSO[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Luciferase and renilla assays were conducted using a dual-luciferase reporter system (Transgene, Beijing, China) according to the manufacturer\u0026rsquo;s protocol. The Firefly and renilla luciferase signals were detected using a microplate reader (Tecan SPARK, Switzerland).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eTime-course analysis\u003c/h2\u003e\u003cp\u003eGene expression data of human primary hepatocyte treated with rosuvastatin for 24 hours or 48 hours were obtained from dataset GSE24188 for time-course analysis. The R (R Core Team 2021) package (mfuzz) (Futschik and Carlisle 2005) to perform time-course analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll quantitative assays were conducted in triplicate. Mfuzz (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mfuzz##sysbiolab##eu\u003c/span\u003e\u003cspan address=\"http://mfuzz##sysbiolab##eu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used for time course analysis. Statistical analyses and graphical representation were performed using GraphPad Prism 8.0.1 (GraphPad Software, San Diego, CA, USA). Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, with \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eESRRA is upregulated in HCC and facilitates cell proliferation in vitro\u003c/h2\u003e\u003cp\u003eTo investigate the potential role of ESRRA in HCC, we firstly explored the expression level of ESRRA in human HCC tissues using data from TGCA database. Analysis of the TCGA database revealed significantly higher \u003cem\u003eESRRA\u003c/em\u003e expression in human HCC tissues compared to normal liver tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Consistently, ESRRA levels were markedly increased in multiple HCC cell lines relative to immortalized normal human hepatocytes (THLE2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, Supplementary Fig.\u0026nbsp;1a). To investigate the functional role of ESRRA, we established ESRRA-knockdown HCC cell models and confirmed the knockdown efficiency at the protein level via western blotting (Supplementary Fig.\u0026nbsp;1b-c). Functional assays demonstrated that ESRRA silencing significantly inhibited HCC cell proliferation, as evidenced by CCK-8 assays, EdU proliferation assays, and colony formation assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-h, Supplementary Fig.\u0026nbsp;1d-e). Moreover, ESRRA knockdown induced G1 phase arrest, further supporting its role in regulating cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei-j). Conversely, ESRRA overexpression (validated by Western blot; Supplementary Fig.\u0026nbsp;1f\u0026ndash;g) promoted HCC cell proliferation as confirmed by colony formation assays, EdU proliferation assays, and CCK-8, (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek-p, Supplementary Fig.\u0026nbsp;1h-i). ESRRA overexpression also facilitated G1/S transition (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eq-s). The above findings underscore the impact of ESRRA in promoting proliferation in HCC cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eESRRA accelerates HCC cell migration in vitro\u003c/h2\u003e\u003cp\u003eTo evaluate the effect of ESRRA on HCC cell migration, we first performed wound-healing assays in ESRRA-knockdown HCC cells. Silencing ESRRA significantly impaired migratory capacity, as evidenced by reduced wound closure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026ndash;c) and fewer invading cells in transwell assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Given the pivotal role of epithelial-mesenchymal transition (EMT) in cancer metastasis[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], we performed western blot analysis to detect the expression of EMT markers in HCC cells treated with ESRRA siRNA. Western blotting revealed that ESRRA knockdown downregulated mesenchymal markers (N-cadherin, vimentin) while upregulating the epithelial marker E-cadherin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Conversely, overexpression of ESRRA significantly enhanced the migratory ability of HCC cells, as evidenced by wound-healing assays and transwell experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef-h). Concurrently, Western blot analysis revealed that ESRRA overexpression promoted the EMT process: it increased the expression of mesenchymal markers (N-cadherin and vimentin) and decreased the expression of the epithelial marker E-cadherin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). These data establish ESRRA as a critical driver of HCC migration and EMT.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eESRRA promotes tumor growth and metastasis in vivo\u003c/h2\u003e\u003cp\u003eTo further confirm the role of ESRRA in the tumor growth and metastasis of HCC, subcutaneous tumor growth models were established in nude mice. Compared to the blank control and empty vector groups, ESRRA overexpression significantly enhanced tumor growth, as evidenced by increased tumor volume and weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c). No significant difference in body weight was observed across the three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Consistently, pharmacological inhibition of ESRRA using XCT790 (an ESRRA antagonist) markedly suppressed tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-g), while no body weight changes were detected among the blank, DMSO, and XCT790-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). To explore the mechanism underlying ESRRA-mediated tumor growth, we performed IHC staining on excised subcutaneous tumors to detect the expression of ESRRA and Ki67 (a well-established marker of cell proliferation). As expected, ESRRA overexpression significantly upregulated Ki67 expression, while XCT790 treatment reduced Ki67 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei-p). This result links ESRRA\u0026rsquo;s pro-tumorigenic effect to enhanced \u003cem\u003ein vivo\u003c/em\u003e cell proliferation\u0026mdash;consistent with our earlier \u003cem\u003ein vitro\u003c/em\u003e observations on HCC cell proliferation. Moreover, metastasis assays demonstrated that ESRRA overexpression enhanced lung metastasis, as reflected by increased number and size of metastatic nodules. In contrast, XCT790 treatment significantly attenuated metastatic dissemination compared to the DMSO control group (Fig. q-t). In summary, our \u003cem\u003ein vivo\u003c/em\u003e data demonstrate that ESRRA functions as a critical regulator of HCC progression by promoting tumor growth and metastasis, while its inhibition effectively suppresses these malignant phenotypes. These findings highlight ESRRA as a potential therapeutic target for HCC intervention.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eESRRA activates PHF5A/PI3K/AKT pathway in HCC cells\u003c/h2\u003e\u003cp\u003eTo unravel the molecular mechanism by which ESRRA drives HCC progression, we performed transcriptomic analysis using RNA-Seq.\u0026nbsp;Volcano plots revealed differentially expressed genes (DEGs) between control and ESRRA-knockdown groups (Supplementary Fig.\u0026nbsp;2a). To identify potential transcriptional targets of ESRRA, we integrated the RNA-Seq data with three independent transcription factor (TF) prediction databases: Gene Transcription Regulation Database (GTRD), hTFtarget, and Cistrome DB (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, Supplementary Fig.\u0026nbsp;2b). Next, we used RT-qPCR to validate the overlapping candidate genes from the RNA-seq and database analyses. Among these candidates, PHF5A showed the most robust and consistent regulation across four HCC cell lines: ESRRA knockdown significantly reduced PHF5A mRNA expression, while ESRRA overexpression markedly increased it (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-c, Supplementary Fig.\u0026nbsp;2c\u0026ndash;f). To confirm this regulation at the protein level, we performed western blotting and IHC staining. Both assays confirmed that PHF5A protein levels closely mirrored its mRNA expression patterns, further supporting ESRRA-dependent regulation of PHF5A (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, Supplementary Fig.\u0026nbsp;2g-m). To assess the clinical relevance of PHF5A in HCC, we analyzed public datasets. Data from the HPA and TGCA databases revealed that PHF5A expression was significantly higher in human HCC tissues compared to normal liver tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee\u0026ndash;f). Critically, HCC patients with high PHF5A expression had significantly shorter 5-year overall survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg), indicating that PHF5A not only correlates with HCC development but also serves as a potential prognostic marker.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe binding sites of ESRRA on \u003cem\u003ePHF5A\u003c/em\u003e promoter were predicted by JASPAR database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://jaspar.genereg.net/\u003c/span\u003e\u003cspan address=\"http://jaspar.genereg.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Supplementary Fig.\u0026nbsp;2n). Notably, the sequence of binding site with the highest score is highly conserved across different host species (Supplementary Fig.\u0026nbsp;2o), suggesting evolutionary conservation of this regulatory interaction. To validate direct binding, we performed luciferase reporter assays. Cells co-transfecting HCC cells with a wild-type (WT) PHF5A promoter reporter plasmid (PHF5A-WT) and an ESRRA-overexpressing plasmid (pcDNA-ESRRA) resulted in significantly higher luciferase activity compared to co-transfection with PHF5A-WT and empty vector (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh-i). To confirm specificity, we generated PHF5A promoter reporter plasmids with mutations at the three predicted ESRRA binding sites. ESRRA overexpression increased luciferase activity only in cells transfected with PHF5A-WT, not in those with mutations at either site 1 or site 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej\u0026ndash;k). These results confirm that ESRRA directly binds to the PHF5A promoter to activate its transcription.\u003c/p\u003e\u003cp\u003eTo explore the downstream pathway of ESRRA, we performed KEGG analysis. The KEGG pathway analysis of differentially expressed genes downstream of ESRRA identified PI3K/AKT signaling as the most significantly enriched pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). The result of KEGG analysis was validated by western blotting. Western blotting validated that ESRRA knockdown suppressed PI3K/AKT phosphorylation, while its overexpression activated this axis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003em, Supplementary Fig.\u0026nbsp;2p-q). Finally, we tested whether PHF5A mediates ESRRA-induced PI3K/AKT activation. PHF5A knockdown significantly suppressed the increased PI3K/AKT phosphorylation triggered by ESRRA overexpression; conversely, PHF5A overexpression rescued the reduced PI3K/AKT phosphorylation caused by ESRRA knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003en, Supplementary Fig.\u0026nbsp;2r-s). These results demonstrate that PHF5A acts as a critical downstream mediator linking ESRRA to PI3K/AKT activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003en, Supplementary Fig.\u0026nbsp;2r-s). Our findings establish ESRRA as a key transcriptional activator of PHF5A, which drives PI3K/AKT signaling activation.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eESRRA promotes HCC through PHF5A dependent pathway\u003c/h2\u003e\u003cp\u003eTo determine whether PHF5A mediates the effect of ESRRA on HCC cells, we performed rescue experiments: we overexpressed PHF5A in ESRRA-knockdown HCC cells and silenced PHF5A in ESRRA-overexpressing (OE) HCC cells, then assessed changes in cell proliferation, cell cycle progression, and migration. Strikingly, the overexpression of \u003cem\u003ePHF5A\u003c/em\u003e partially reversed the diminished proliferation capacity and the arrested G1 phrases in ESRRA-knocked down cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-d, Supplementary Fig.\u0026nbsp;3a-b). The silence of \u003cem\u003ePHF5A\u003c/em\u003e reversed the increased proliferation of HCC cells induced by ESRRA overexpression and facilitated G1/S transition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee-h, Supplementary Fig.\u0026nbsp;3c-d). The overexpression of PHF5A reversed the impaired migration and reduced EMT markers caused by ESRRA knockdown. On the other hand, PHF5A knockdown suppressed the enhanced migration and elevated EMT markers induced by ESRRA overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei-o, Supplementary Fig.\u0026nbsp;3e-h). Taken together, these rescue experiments provide direct evidence that ESRRA promotes HCC malignant behaviors\u0026mdash;including proliferation, G1/S transition, migration, and EMT\u0026mdash;through a PHF5A-dependent pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eESRRA promotes HCC development via PHF5A/PI3K/AKT pathway\u003c/h2\u003e\u003cp\u003eTo confirm that the PI3K/AKT pathway is functionally indispensable for ESRRA-mediated promotion of HCC cell proliferation and migration, we performed pharmacologic intervention experiments using the specific PI3K agonist 740 Y-P and the selective PI3K inhibitor LY294002. The proliferation suppression and G1 arrest induced by ESRRA knockdown were effectively rescued by 740Y-P (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-b). Conversely, the pro-proliferative effect of ESRRA overexpression was abolished by LY294002, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec-d. Similarly, 740Y-P reversed the migration suppression caused by ESRRA knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee-j), whereas LY294002 mitigated the enhanced migration due to ESRRA overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ek-p). These results strongly suggest that the PI3K/AKT pathway plays a crucial role in the ability of ESRRA to promote HCC cell proliferation and migration, further supporting the hypothesis that ESRRA promotes HCC development via the PHF5A/PI3K/AKT pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eStatin inhibits HCC via ESRRA suppression\u003c/h2\u003e\u003cp\u003eGiven that previous studies have demonstrated that cholesterol binds to the ligand-binding domain (LBD) of ESRRA to activate its transcriptional activity, and that statins can reverse this cholesterol-mediated activation[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], we next investigated whether statins inhibit HCC by suppressing ESRRA function. Among clinically used statins, rosuvastatin was selected for subsequent experiments due to its relatively low liver toxicity[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Firstly, we confirmed that rosuvastatin effectively decreased cholesterol levels in HCC cells (Supplementary Fig.\u0026nbsp;4a). A time-course transcriptome was performed using dataset GSE24188.We found that in liver cells, the trend of ESRRA mRNA level belonged to cluster 1, which gradually decreased with prolonged rosuvastatin intervention (Supplementary Fig.\u0026nbsp;4b). RT-qPCR further confirmed that in HCC cells, the mRNA levels of ESRRA declined with extended rosuvastatin treatment, and this effect could be reversed by cholesterol supplementation (Supplementary Fig.\u0026nbsp;4c-d). Dual-luciferase reporter assays indicated that rosuvastatin treatment markedly downregulated ESRRA transcriptional activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-b). Since statins inhibit cholesterol biosynthesis by targeting the endogenous mevalonate pathway[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], we performed a rescue experiment by adding exogenous mevalonate to rosuvastatin-treated HCC cells. As expected, mevalonate supplementation reversed the rosuvastatin-induced reduction in ESRRA mRNA levels and transcriptional activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-b). Consistent with these findings, rosuvastatin also downregulated the mRNA level of \u003cem\u003ePHF5A\u003c/em\u003e, and this effect was also reversed by mevalonate (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec-d), confirming that statins suppress ESRRA activity via mevalonate pathway inhibition.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo confirm that ESRRA mediates the anti-tumor effects of rosuvastatin, we assessed cell proliferation and migration in rosuvastatin-treated cells with or without ESRRA overexpression. ESRRA overexpression completely abolished the anti-proliferative and anti-migratory effects of rosuvastatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee-j, Supplementary Fig.\u0026nbsp;4e-g). Furthermore, mevalonate treatment, which replenished intracellular cholesterol, reversed the anti-proliferation and anti-migratory effects of rosuvastatin in HCC cells. However, this rescue was absent when ESRRA expression was downregulated by XCT790 (an ESRRA-specific antagonist), indicating that the anti-HCC effect of rosuvastatin relied on ESRRA suppression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ek-p, Supplementary Fig.\u0026nbsp;4h-j). The results above further solidify ESRRA as the key mediator of rosuvastatin\u0026rsquo;s anti-HCC activity.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present research, we discover that ESRRA was upregulated in HCC and promotes HCC cell proliferation and migration both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Mechanistically, ESRRA upregulated PHF5A by directly binding to the promoter, which in turn activated PI3K/AKT pathway. Furthermore, we found that rosuvastatin effectively suppresses the expression and transcriptional activity of ESRRA, which contributes to its anti-tumor effects in HCC cells. Our findings not only identify the ESRRA-PHF5A axis as a potential therapeutic target for HCC but also uncover a novel anti-tumor mechanism of statins in HCC progression, providing a molecular rationale for the clinical application of statins in HCC treatment.\u003c/p\u003e\u003cp\u003eThe role of ESRRA varies across tumor types, exhibiting both oncogenic and tumor-suppressive functions depending on the cellular context. While it typically promotes tumorigenesis in prostate, ovarian, and esophageal cancers, it paradoxically suppresses tumor growth in triple-negative breast cancer, underscoring the importance of context-dependent regulation[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the present study, we demonstrate that ESRRA functioned as a promoter in HCC proliferation and migration. Intriguingly, this pro-tumorigenic role appears to contradict a previous report showing that ESRRA -null mice exhibit increased susceptibility to liver carcinogenesis. ESRRA deficiency in hepatocytes induces necrosis-driven compensatory proliferation, while its loss in Kupffer cells hyperactivates NF-κB signaling, exacerbating inflammation and tumor initiation[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Together, these seemingly contradictory observations highlight a stage-dependent dual role of ESRRA in HCC pathogenesis. Specifically, during the pre-malignant stages of liver diseases, ESRRA may function as a tumor-suppressive factor by limiting hepatocyte necrosis and suppressing Kupffer cell-mediated inflammation, thereby reducing tumor initiation risk[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In established HCC, however, our data indicate that ESRRA switches to an oncogenic role, driving malignant hepatocyte proliferation and migration. This dual regulatory mechanism highlights the complex, context-specific functions of ESRRA in liver cancer development and progression.\u003c/p\u003e\u003cp\u003eFunctioning as a transcription factor, ESRRA exerts its effects by regulating downstream target genes. Our RNA-sequencing analysis identified PHF5A \u0026mdash;a critical component of the SF3b spliceosomal complex\u0026mdash;as a direct transcriptional target of ESRRA in HCC. PHF5A governs alternative splicing, a process frequently dysregulated in cancer[\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Notably, PHF5A is overexpressed in multiple cancers and promotes proliferation and migration[\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, its role in HCC - particularly its regulatory mechanisms and proliferative effects, remained incompletely understood[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Our study confirmed that ESRRA directly promotes PHF5A transcription in HCC cells, and further demonstrated that PHF5A mediates ESRRA-driven HCC cell proliferation and migration. This discovery thereby uncovers a novel regulatory mechanism responsible for PHF5A upregulation in HCC. While no PHF5A-specific inhibitors currently exist, XCT790 \u0026mdash;a well-characterized ESRRA antagonist\u0026mdash;has demonstrated efficacy in preclinical models, offering a translational avenue for targeting this axis. Collectively, our study elucidates the upstream regulatory mechanism governing PHF5A upregulation in HCC, which may offer a promising therapeutic strategy for HCC patients with elevated PHF5A expression.\u003c/p\u003e\u003cp\u003eThe PI3K/Akt pathway is a key signaling pathway driving survival of cancer, including HCC. Its activation promotes HCC cell cycle progression[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], inhibits cell apoptosis[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], facilitates angiogenesis and EMT [\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and induces drug resistance[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Our data provide evidence that ESRRA promotes cell proliferation and migration in HCC through activating PI3K/AKT pathway. The present study uncovers ESRRA as a novel upstream regulator that activates this cascade, and highlight ESRRA's significance as a potential new therapeutic target for the aberrant activation of the PI3K/AKT pathway in HCC. Furthermore, activation of the PI3K/AKT pathway is a key factor in the resistance to first - line chemotherapeutic agents such as sorafenib and Lenvatinib, which is a major challenge in HCC treatment[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Therefore, combining ESRRA inhibitors may relieve sorafenib resistance and enhance the therapeutic efficacy of HCC treatment.\u003c/p\u003e\u003cp\u003eMetabolic reprogramming is a hallmark of cancer[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Clinical evidence has demonstrated that metabolism-modulating drugs, including metformin and SGLT2 inhibitors, significantly improve the prognosis of HCC patients, highlighting the pivotal role of metabolic regulation in HCC treatment[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Alongside glucose metabolism, lipid metabolism has emerged as a critical regulator [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Cholesterol supplies lipids for cell membrane fluidity, signaling transduction, and ECM adhesion, all of which contribute to tumor proliferation and migration in HCC[\u003cspan additionalcitationids=\"CR50 CR51\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], while conflicting evidence exists that elevated serum cholesterol enhances natural killer cell-mediated anti-tumor activity and reduces liver tumor growth in mice[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. This discrepancy highlights that intracellular cholesterol, rather than extracellular cholesterol, functions as a promoter of HCC progression. Statins, which lower intracellular cholesterol by inhibiting de novo synthesis, thus represent a plausible therapeutic strategy. Clinical studies have confirmed that statins not only reduce HCC incidence risk but also improve HCC patients\u0026rsquo; prognosis[\u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Furthermore, unlike glucose-lowering drugs-which exert anti-HCC effects primarily in patients with diabetes mellitus, statins can elicit such effects even in individuals without metabolic disorders. Consistent with previous studies, we found that rosuvastatin not only effectively reduced intracellular cholesterol levels in HCC cells but also suppressed their proliferation and migration. Nevertheless, the exact molecular mechanisms of statins' anti - HCC effects remain to be fully elucidated.\u003c/p\u003e\u003cp\u003ePrevious studies have shown that cholesterol can upregulate ESRRA expression[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], or act as a ligand for ESRRA to enhance its transcriptional activity[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], implying a potential association among cholesterol, statins and ESRRA in HCC. In this study, we found that rosuvastatin can simultaneously suppress both ESRRA expression and its transcriptional activity. This dual-inhibitory effect can be reversed by intracellular cholesterol repletion. Moreover, the anti-HCC effects of rosuvastatin were ESRRA-dependent and could be also reversed by cholesterol replenishment. These results demonstrated that rosuvastatin suppresses HCC by targeting cholesterol - dependent ESRRA activation. To our knowledge, this is the first study to provide mechanistic evidence linking ESRRA inhibition to the anti-HCC effects of statins. Clinically, while several trials have reported that statins have a favorable safety profile and are associated with reduced HCC recurrence and mortality[\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], another study failed to observe significant therapeutic benefits[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Therefore, additional clinical research is needed to explore whether statin efficacy in HCC correlates with ESRRA expression levels in patients\u0026rsquo; tumor tissues.\u003c/p\u003e\u003cp\u003eAlthough we have established the activation of the ESRRA -PHF5A-PI3K/AKT axis, the underlying mechanism by which PHF5A triggers the PI3K/AKT pathway in HCC remains elusive. Previous studies have indicated that PHF5A may regulate downstream genes through alternative splicing[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], which might potentially activate this signaling pathway. Therefore, future studies are warranted to explore the detailed interactions between PHF5A and the PI3K/AKT pathway. In addition, while statins have been shown to indirectly suppress ESRRA in the present study, the presence of a ligand - binding domain in ESRRA implies the feasibility of directly targeting it with small - molecule compounds[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Future work could employ molecular simulations and docking to screen compounds that modulate ESRRA activity.\u003c/p\u003e\u003cp\u003eIn summary, our study has significantly advanced the understanding of the role of ESRRA in HCC, elucidating a novel pathway involving ESRRA, PHF5A and the PI3K/AKT signaling cascade. Specifically, we demonstrated that ESRRA was upregulated in HCC, contributing to its progression via the PHF5A/PI3K/AKT signaling pathway. Moreover, we have established that ESRRA serves as a crucial mediator in the anti - tumor effects of rosuvastatin. As a nuclear receptor, ESRRA can be ligand activated and, therefore, holds great promise as a potential drug target in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFundings\u003c/h2\u003e\u003cp\u003eThis study was supported by 5010 Cultivation Program of Clinical Research of Sun Yat-Sen University (Grant number: 04A005001000015) and horizontal foundation of Sun Yat-Sen University (Grant number: 06A001001000434).\u003c/p\u003e\u003cp\u003eCompeting interests\u003c/p\u003e\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\u003cp\u003eJiao Gong, Yuankai Wu, Yusheng Jie, and Yutian Chong designed this study and revised the manuscript. Zhiping Wan and Shuying Huang analyzed and interpreted the data. Xiang Cai carried out experiments and wrote the manuscript. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eOriginal data are available upon reasonable request from the corresponding author.\u003c/p\u003e\u003cp\u003eEthical approval\u003c/p\u003e\u003cp\u003e All animal experiments were approved by the Animal Ethics Committee (approval number: IACUC \u0026minus;\u0026thinsp;202505166).\u003c/p\u003e\u003cp\u003eConsent for publication\u003c/p\u003e\u003cp\u003eAll authors approved the publication of this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F et al (2024) Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 74(3):229\u0026ndash;263\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGigu\u0026egrave;re V (1999) Orphan nuclear receptors: from gene to function. 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Mol Cell 74(6):1250\u0026ndash;1263 .e6\u003c/span\u003e\u003c/li\u003e\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":"ESRRA, PHF5A, hepatocellular carcinoma, statin, cholesterol","lastPublishedDoi":"10.21203/rs.3.rs-7867831/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7867831/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe estrogen related receptor alpha (ESRRA), a key member of the estrogen receptor-related receptor (ERR) family, has been extensively implicated in tumor progression across multiple cancers. Existing studies highlight its pivotal role in cancer cell proliferation and migration. However, its specific function and underlying molecular mechanisms in hepatocellular carcinoma (HCC) remain incompletely understood.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eThe effects of ESRRA on HCC cells proliferation and migration were investigated \u003cem\u003ein vitro\u003c/em\u003e (CCK-8, colony formation, EdU proliferation, wound-healing, transwell assays, and Epithelial-mesenchymal transition (EMT) marker analysis) and \u003cem\u003ein vivo\u003c/em\u003e (Balb/c nude mouse subcutaneous xenograft and lung metastasis models). RNA sequencing and dual-luciferase reporter assays were employed to identify ESRRA\u0026rsquo;s downstream targets and pathways.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eESRRA promoted HCC cell proliferation and migration \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Mechanistically, ESRRA transcriptionally upregulated PHD finger protein 5A (PHF5A), which subsequently activated the PI3K/AKT signaling cascade. The cholesterol-lowering drug rosuvastatin exerted anti-tumor effects on HCC by downregulating ESRRA and, meanwhile, suppressing its transcriptional activity through depleting intracellular cholesterol.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eESRRA promotes HCC progression via the PHF5A /PI3K/AKT axis and mediates rosuvastatin's anti-tumor effect. Targeting ESRRA-PHF5A may be a therapeutic strategy for HCC.\u003c/p\u003e","manuscriptTitle":"ESRRA-Driven PHF5A Activation Promotes Hepatocellular Carcinoma Progression and is Therapeutically Targeted by Rosuvastatin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-06 12:43:14","doi":"10.21203/rs.3.rs-7867831/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":"3afc5fb9-1af0-4bb2-8154-f0a3f4eca995","owner":[],"postedDate":"November 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-05T05:45:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-06 12:43:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7867831","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7867831","identity":"rs-7867831","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}