FOXO6 specifically mediates overactivation of Rac1 in hepatocellular carcinoma

FOXO6 specifically mediates overactivation of Rac1 in hepatocellular carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article FOXO6 specifically mediates overactivation of Rac1 in hepatocellular carcinoma Jingfang Diao, Bo Xie, Qing Ye, Shunjun Fu, Xuewen Liu, Junming He, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3782217/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 Rac1 activation is a common occurrence in various tumors and is often associated with poor prognosis, underscoring the potential therapeutic value of targeting the Rac1 pathway. Therefore, selectively inhibiting the heightened Rac1 activity in tumor cells may represent an innovative approach to cancer treatment. In this study, we found the increase in Rac1 expression contributes to heightened Rac1 activity and enhanced migration of HCC cells. Notably, our investigations identified FOXO6, rather than HIF-1α, Smad7, miR-142-3p, or miR-137, as the mediator of Rac1 expression. FOXO6 exhibits transcriptional activation and correlates with the early recurrence of HCC following hepatectomy. The transcriptional activation of the Rac1 gene hinges on a FOXO-binding sequence in the Rac1 gene promoter. FOXO6 was found to directly bind to this sequence both in vitro and in vivo . Ultimately, Rac1 operates downstream of the FOXO6-dependent pro-migration signaling cascade. Our findings illuminate the direct role of FOXO6 in mediating the upregulation of Rac1 expression and activity in HCC cells. This discovery unveils a differentially activated FOXO6/Rac1 pathway in liver cancer, thereby positioning FOXO6 as a potential therapeutic target for liver cancer treatment, offering the prospect of mitigating excessive side effects. Hepatocellular carcinoma Rac1 FOXO6 target gene transcriptional regulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Rac1 has long been recognized for its pivotal role in the development and metastasis of liver cancer. Notably, the Rac1 protein exhibits heightened expression in invasive hepatocellular carcinoma (HCC) cell lines, and its functionality is closely associated with cell motility and the aggregation of cytoskeletal components 1 . Inhibiting Rac1 activity has been demonstrated to trigger microfilament depolymerization, consequently diminishing cell motility 2,3 . Importantly, Rac1 has been identified as a key player in the growth of primary HCC tumors and the progression of HCC metastasis 4 . As of now, Rac1 is regarded as a promising target for curtailing the acquired and intrinsic resistance of liver tumors and their metastatic potential 5 . Furthermore, it is worth noting that Rac1 overexpression has been linked to the development and advancement of gastric cancer, testicular cancer, and breast cancer 6–8 . Nevertheless, it is imperative to acknowledge that Rac1 is ubiquitously expressed across various tissues, and any disruption in Rac1 signaling may yield deleterious effects. This is primarily due to the involvement of Rac1-driven cellular processes in numerous physiological and pathological conditions. These encompass actin cytoskeletal reorganization, cell transformation, induction of DNA synthesis, superoxide production, axonal guidance, cell migration, neuronal polarization, cancer, cardiovascular disease, neurodegenerative disease, pathological inflammatory responses, kidney disease, and infectious disease 1,9–11 . Hence, it may be prudent to prioritize the identification of factors specifically responsible for elevating Rac1 activity as potential drug targets, as opposed to directly inhibiting Rac1 activity itself. Forkhead box protein O6 (FOXO6), a member of the forkhead family, has remained relatively enigmatic with regard to its involvement in tumorigenesis. Nonetheless, recent investigations have shed some light on its relevance. FOXO6 expression has been observed in HCC tissues, correlating with oxidative stress levels 12 . Additionally, overexpression of FOXO6 has been found to promote tumorigenicity in gastric cancer cells 13 , and it is highly upregulated in both breast cancer and colorectal cancer tissues 14,15 . In adult physiology, FOXO6 gene expression is primarily confined to the endopiriform nuclei and hippocampus of the brain 16 . Nonetheless, the precise mechanisms underlying FOXO6's role in carcinogenesis and progression remain poorly understood. In the present study, we have substantiated the up-regulation of both FOXO6 and Rac1 expression in HCC tissues and elucidated their involvement in HCC cell migration. Our findings unequivocally establish Rac1 as a direct target gene of FOXO6 in HCC cells, positioning Rac1 as a downstream component of the FOXO6-dependent pro-migration signaling cascade. These revelations underscore the potential of FOXO6 as a therapeutic target for patients grappling with liver cancer. MATERIALS AND METHODS Cell culture Cells were cultured as described in our previous study 17,18 . Human normal hepatic cell lines (THLE-2 and THLE-3) and human HCC cell lines (SNU-182, SNU-475 and HepG2) were all purchased from American Type Culture Collection (ATCC). All cell lines used, were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 100 U/ml penicillin-streptomycin (Gibco) in a 5% CO2 humidified incubator at 37°C. HUVEC was cultured in LSGS-supplemented Medium 200 (Gibco) with 10% FBS and 100 U/ml penicillin-streptomycin. Patient tissue specimens The samples were collected between 2015 and 2023 at Guangdong Provincial Hospital of Traditional Chinese Medicine (Guangzhou, China). The HCC cases selected were based on a clear pathological diagnosis after surgery and follow-up data and had not received neoadjuvant chemotherapy or radiotherapy. HCC tissues and matched tumor-adjacent morphologically normal liver epithelial tissues were frozen and stored in liquid nitrogen. The collected HCC tissues and adjacent normal liver tissues used in this study were in accordance with the ethical standards of the institute research ethics committee of Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou University of Chinese Medicine (Reference No. ZE2023-304-01) and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all participants before the procedure. Immunohistochemical chemistry (IHC) analyzes Tissues were cut into 5-µm sections and processed for IHC using a standard two-step technique as demonstrated previously. Alkaline phosphatase substrate kit III was obtained from Vector Laboratories, Inc. Anti-mouse alkaline phosphatase conjugate (Sigma) was applied to each section for 1 h at room temperature, sections were washed, and alkaline phosphatase substrate was applied for 20 min. Before counterstaining and dehydration, the sections were then subjected to IHC for different antibodies as the chromagen. Sections were washed and counterstained with Mayer’s hemalum. Microscopy was performed using an Olympus Fluoview confocal microscope or an Olympus Provis microscope. Immunofluorescence analyzes Tissues were cut into 5 µm sections and processed for immunofluorescence using a standard two-step technique. Tissue sections were washed twice with PBS and followed by permeabilization with 0.1% Triton X-100 in TBS and blocking in 3% donkey serum. Tissue sections were then washed twice in PBS and treated by cold blocking buffer for 1h. After sequential treatment with NH 4 Cl (50 mM in 20 mM glycine) for 10 min, the indicated antibody (1:100 in bovine serum albumin) was added and incubated overnight at 4°C. After an additional incubation for 1 h at room temperature with Hoechst 33258 and Alexa Fluor® 488 -conjugated secondary antibody (Abcam) (1:400 in bovine serum albumin), the slides were mounted in anti-fading solution (Permafluor, Beckman Coulter, Krefeld, Germany) and stored at 4°C, followed by confocal laser-scanning microscopy. Transwell migration assay Cells were plated at 1 × 10 5 cells/well in 8.0-mm pore-sized 24-well Transwell inserts (Corning) with serum-free RPMI-1640, and the lower chamber was filled with 0.6 ml/well of RPMI-1640 supplemented with 2% FBS. The cells were incubated at 37°C for 24h. Thelower side of the filter was fixed in 4% paraformaldehyde (Electron Microscopy Science) and stained with 0.5% Crystal violet (Sigma-Aldrich) for 20 min, followed by examination under a microscope whereby 5 randomly non-overlapping high-power fields (HPFs, ×200) were selected to count the stained migrated cells. All the experiments in each group were performed in triplicate. Rac1 activity assay The activity of Rac1 (GTP bound form) was assayed in the ovarian protein extract using G-LISA Rac1 activation assay Biochem Kit, as per the manufacturer’s instructions and that is already validated. Briefly, a total of 50 µg protein extract was added to each corresponding well pre-coated with Rac-GTP-binding protein. This was incubated at 4°C for 30 min followed by successive incubation with 50 µl of anti-Rac1 for 45 min. Later, secondary antibody conjugated with HRP (50 µl) was incubated for 45 minutes. Subsequently, 50 µl of HRP detection reagent was added to each well, followed by incubation for another 20 min. The reaction was stopped by the addition of 50 µl of HRP stop solution and the absorbance was recorded at 490 nm. Real-Time Polymerase Chain Reaction (Real-time PCR) Total RNA was extracted using the EastepTM Universal RNA Extraction Kit (Promega, Madison, WI) and transversely transcribed with the GoScriptTM Reverse Transcription System (Promega, Madison, WI) according to the manufacturer’s instruction. Real-time PCR was performed in triplicate with GoTaq® qPCR Master Mix (Promega, Madison, WI) and run on the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Framingham, MA). Western blotting Western blotting analysis was performed as described in our previous study 17,18 . Briefly, the protein extracts from cells or tissues were separated by 8% or 10% SDS‒PAGE, transferred to a PVDF membrane and incubated with the indicated primary antibody. The signal was detected by an ECL detection system (GE Healthcare). siRNA interference RNA interference analysis was performed as described in our previous study. Specific siRNAs were from Dharmacon (ONTARGETplus SMARTpool) and Santacruz; the negative control (NC) siRNA (no silencing small RNA fragment) was synthesized by GenChem Co. (Shanghai, China). siRNAs were transfected into cells using Lipofectamine RNAiMAX (iMAX) (Life Technologies, InvitrogenTM, Cat No: #13778150). Co-immunoprecipitation Co-immunoprecipitation was performed as described previously 19 . For in vivo protein interaction, cell extracts were prepared by solubilizing 10 7 cells in 1 ml of cell lysis buffer made of 1% Triton X-100, 150 mM NaCl, 20 mM Tris-Cl at pH 7.4, 1 mM EDTA, 1 mM EGTA, 1 mM Na 3 VO 4 , 2.5 mM pyrophosphate, 1 mM glycerol phosphate and protease inhibitor mixture for 10 min at 4°C. After brief sonication, the lysates were cleared by centrifugation at 15,000 × g for 10 min at 4°C, and the cell extract was immunoprecipitated with 2 µg of antibodies and incubated with 100 µl of protein G plus protein A-agarose for 12 h at 4°C by continuous inversion. Immunocomplexes were pelleted, washed 3 times, boiled in Laemmli buffer and analyzed by Western blotting. Dual-luciferase reporter assays A dual-luciferase reporter assay was performed as described in our previous study 20 . Constructs were transfected into cells using Lipofectamine 2000. For the dual-luciferase reporter assays, cells were transfected with 1 µg of a luciferase reporter plasmid and 200 ng of the pRL-CMV Renilla luciferase reporter plasmid (Promega). After transfection, cells were kept in conditioned media for 12 or 24 h and then transferred to treatment media for 12 h. Firefly luciferase activity was normalized to Renilla luciferase activity according to the protocol. Chromatin immunoprecipitation assays (ChIP) ChIP was performed as described in our previous studies 20 . Briefly, ChIP assays were performed using the ChIP assay kit (Upstate Cell Signaling Solutions) according to the manufacturer’s instructions. Egr-1 antibody (#4153, Cell Signaling Technology) was used for immunoprecipitation. Purified DNA was subjected to PCR amplification using primers spanning the Rac1 gene promoter FOXO site. Statistical analysis All statistical analyzes were performed using PASW Statistics 22.0 (SPSS Inc., Chicago, IL), with the exception of the significance in bar graphs, in which case analyzes were performed by applying the independent t test using Microsoft Office Excel software (Microsoft Corp., Redmond, WA). The statistics in the graphs represent the means ± S.E.s. Bars represent at least three independent experiments. A p value of less than 0.05 was considered significant. Ethical statement This study were approved by ethical standards of the institute research ethics committee of Guangdong Provincial Hospital of Traditional Chinese Medicine，Guangzhou University of Chinese Medicine (Reference No. ZE2023-304-01) and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all participants before the procedure. Results Upregulation of Rac1 expression contributes to HCC cell migration It has been established that Rac1 can facilitate the migration of HCC cells. In our study, we observed a significant elevation in Rac1 mRNA and protein expression levels in human HCC cell lines compared to human normal hepatic cell (NHC) lines (Fig. 1A and 1B). Additionally, we confirmed a marked upregulation of Rac1 in HCC tissues in comparison to adjacent normal liver tissues (Fig. 1C). To substantiate the role of Rac1 in promoting HCC cell migration, we employed specific siRNAs to partially inhibit Rac1 upregulation, and our findings confirmed the significance of Rac1 in this process (Fig. 1D). To assess whether Rac1 activity was indeed heightened in HCC cells, we conducted a comparative analysis of total Rac1 activity and relative Rac1 activity between HCC cells and NHC cells. Interestingly, we observed an increase in the total activity of Rac1 in HCC cells (Fig. 1E). However, the relative activities of Rac1 remained unaltered (Fig. 1F). Furthermore, the inhibition of Rac1 expression prevented the escalation of total Rac1 activity without affecting the relative activity of Rac1 (Fig. 1G). These findings collectively demonstrate that the upregulation of Rac1 expression contributes to the activation of Rac1 and, consequently, the migration of HCC cells. Expression upregulation of Rac1 is not mediated by HIF-1α, Smad7, miR-142-3p or miR-137 In HCC, previous reports have indicated an elevation in Rac1 expression 2 , 21 . To substantiate the transcriptional upregulation of the Rac1 gene, we transfected cells with a luciferase reporter construct containing the Rac1 gene promoter sequence (nucleotides +2825 to -584), referred to as Rac1-LucR. Our observations revealed an approximately fourfold increase in Rac1-LucR activity in HCC cells when compared to NHC cells (Fig. 2A), thereby confirming the transcriptional upregulation of Rac1 in HCC cells. Smad7, a pivotal inhibitor of the TGFβ-Smad signaling pathway, has the capacity to augment Rac1 promoter activity by attenuating CtBP1 binding to the mouse Rac1 promoter. This effect has been observed in both mouse oral mucositis and human oral keratinocytes 22 . However, our investigation uncovered a downregulation of Smad7 in human HCC tissues and cell lines relative to adjacent normal tissues and NHC lines (Fig. 2B and 2C). Furthermore, the phosphorylation and nuclear localization of Ser-249-Smad7, which influence the transcriptional activity of Smad7 23 , exhibited a decrease in human HCC tissues compared to adjacent normal tissues (Fig. 2D). Notably, the use of Smad7 siRNAs did not exert any influence on the promoter activity, mRNA levels, or protein levels of Rac1 (Fig. 2E-2G), thus confirming that Smad7 does not function as a regulator of Rac1 expression in HCC cells. Previous studies have proposed regulatory roles for HIF-1α, miR-142-3p, or miR-137 in modulating Rac1 expression 2 , 22 , 24 , 25 . However, our experiments employing HIF-1α, miR-142-3p, or miR-137 siRNAs revealed no discernible impact on Rac1 promoter activity, mRNA levels, or protein levels (Fig. 2E-2G). This underscores an HIF-1α, miR-142-3p, or miR-137-independent mechanism governing Rac1 expression in HCC cells. Collectively, these findings suggest that the upregulation of Rac1 expression in HCC cells is not mediated by HIF-1α, Smad7, miR-142-3p, or miR-137. The FOXO binding site is required for Rac1 promoter activation To ascertain the transcription factor responsible for regulating the activity of the Rac1 gene (Gene ID: 5879) promoter 26 , we conducted an investigation to pinpoint the core sequences essential for promoter activation. We designed a series of reporter constructs featuring deletions of the 5'-flanking region of the Rac1 promoter, all of which retained the same 3' end. Among these, one deletion construct (encompassing nucleotides -231 to +455) exhibited a response analogous to that of the original construct (encompassing nucleotides -1918 to +455) in HCC cells. In contrast, another deletion construct (encompassing nucleotides +16 to +455) effectively abolished the heightened promoter activity (Fig. 3A). Consequently, we delineated the principal Rac1 transcriptional control element to the region spanning nucleotides -231 to +16. Promoter analysis unveiled the presence of a typical Egr-1 binding site situated between nucleotides -31/-23 (5'-CGCCGCCGC-3' or 3'-GCGGCGGCG-5') in the core region of the Rac1 promoter (Fig. 3B). Notably, this putative Egr-1 site coincided with its classical binding site in the PTEN promoter 27 . To assess the role of this putative Egr-1 site in Rac1 promoter activation, we introduced mutations within the site into the reporter vector. Specifically, we created a human mutant Egr-1 binding site Rac1 reporter gene designated as Rac1-LucR-MtEgr-1 (Fig. 3C). Intriguingly, our results revealed that these mutations did not exert any influence on the activity of the Rac1 promoter in HCC cells (Fig. 3D). Furthermore, our experiments involving Egr-1 siRNAs demonstrated their inability to impact the promoter activity of Rac1 (Fig. 3E), thus confirming that Egr-1 does not serve as a regulator of Rac1 expression in HCC cells. Additionally, our promoter analysis unveiled a potential binding site for Forkhead-box O subclass proteins (FOXO) between nucleotides -208/-202 (5'-TTTCC TGTTTCT CTGC-3' or 3'-GCAG AGAAACA GGAAA-5') in the core region of the Rac1 promoter (Fig. 3B). Notably, this putative FOXO site contains the core sequence recognized by FOXO proteins 28 . To assess the role of this putative FOXO binding site in Rac1 promoter activation, we introduced mutations within the site into the reporter vector, generating a human mutant FOXO site Rac1 reporter gene referred to as Rac1-LucR-MtFOXO (Fig. 3C). Our results unequivocally demonstrated that these mutations disrupted the activation of the Rac1 promoter in HCC cells (Fig. 3D). These findings conclusively establish the significance of the putative FOXO site (nucleotides -208 to -202) in Rac1 promoter activation in HCC cells. FOXO6 mediates the expression of Rac1 The "O" subclass of the human FOX family encompasses four members (FOXO1, FOXO3, FOXO4, and FOXO6), each contributing to the control of various metabolic processes, cell survival, cellular proliferation, DNA damage repair responses, and stress resistance 29 . As transcription factors, the nuclear localization of FOXO proteins is a fundamental prerequisite for the regulation of their target gene expression. Previous studies conducted in mammalian cell lines have established that when Akt phosphorylates FOXO1, FOXO3, and FOXO4, these proteins relocate from the nucleus to the cytosol 30 . In our current study, we observed no difference in the phosphorylation of FOXO1 (Thr24), FOXO3 (Thr32), FOXO4 (Ser197), or their upstream kinase Akt (Ser473) when comparing human HCC tissues and cell lines with adjacent normal tissues and NHC lines. This observation held true despite a significant elevation in Rac1 protein levels being evident in human HCC tissues and HCC cell lines (Fig. 4A). Intriguingly, we noted that FOXO1, FOXO3, and FOXO4 predominantly exhibited cytosolic localization in both HCC and NHC cells. In stark contrast, FOXO6 primarily resided in the nucleus of HCC cells when compared to NHC cells (Fig. 4B). To further elucidate which FOXO member is involved in the regulation of Rac1 expression, we employed specific siRNAs targeting FOXO members. Our findings indicated that only siRNAs targeting FOXO6 had a discernible impact on the promoter activity, mRNA levels, and protein levels of Rac1 (Fig. 4C-4E). This underscores a FOXO6-dependent mechanism governing Rac1 expression regulation. Transcriptional activation of FOXO6 is associated with the early recurrence of HCC after hepatectomy Next, we embarked on an investigation to assess the correlation between FOXO6 expression and the prognosis of HCC. A prior study had already reported the overexpression of FOXO6 in HCC tissues, thus substantiating its relevance in the pathogenesis of HCC 12 . Our own analysis reaffirmed these findings, as we observed a marked upregulation of FOXO6 protein levels in HCC tissues in comparison to adjacent normal liver tissues (Fig. 5A). Furthermore, the protein levels of FOXO6 were significantly higher in HCC cells compared to NHCs (Fig. 5B). To further elucidate the clinical implications of FOXO6 expression, we conducted a retrospective analysis in 37 patients who experienced HCC recurrence following hepatectomy. This analysis revealed a close association between FOXO6 overexpression and vascular invasion (P=0.007) as well as histological differentiation (P=0.04). However, no significant correlations were established between FOXO6 overexpression and parameters such as tumor, node, metastasis (TNM) stages, tumor diameter, tumor number, and serum AFP levels (Table 1). Subsequent analyses focused on the average duration of HCC recurrence in these 37 patients, which was found to be 17.1 months on average (ranging from 4 to 35 months) post-hepatectomy. Kaplan-Meier survival curves were employed for further assessment, revealing that patients devoid of FOXO6 overexpression experienced significantly longer recurrence-free survival in comparison to those with FOXO6 overexpression (P=0.024, log-rank test) (Fig. 5C). In summary, our recurrence-free survival analysis establishes a significant correlation between increased FOXO6 expression and an adverse prognosis among HCC patients following hepatectomy. Transcriptional activation of FOXO6 directly activates Rac1 gene A previous study has highlighted that FOXO6's transcriptional activity is subject to regulation not solely based on nuclear localization but also on the phosphorylation status of Ser184 31 . Specifically, Ser184-phosphorylated FOXO6 is known to interact with 14-3-3β, a scaffold protein, resulting in the sequestration of FOXO6 and its prevention from binding to target promoters 32 , 33 . To investigate whether FOXO6 is indeed activated in HCC tissues and cultured cells, we conducted an immunodepletion study. Our findings indicated that the immunodepletion of Ser184-phosphorylated FOXO6 had only a minor impact on the total FOXO6 levels, with reductions of 16.5% in HCC tissues and 15.8% in SNU-475 cells (Fig. 6A). Thus, it can be inferred that FOXO6 appears to be activated in both HCC tissues and cell lines. In order to ascertain whether FOXO6 interacts directly with the Rac1 promoter, both in vitro and in vivo , we employed a Chromatin Immunoprecipitation (ChIP) assay. Precipitated immunocomplexes were subjected to Western blot analysis using an anti-FOXO6 antibody. The results revealed the presence of FOXO6 protein cross-linked to DNA in HCC cells and tissues (Fig. 6B). Subsequently, the precipitated DNA was subjected to PCR using primers flanking the FOXO6 binding site in the Rac1 promoter. This assay led to a robust amplification of the DNA fragment from both Rac1-treated cells and mice (Fig. 6C). Thus, our findings affirm that FOXO6 directly binds to the FOX binding site region in the Rac1 promoter, thereby promoting the expression of Rac1. Rac1 acts downstream to mediate the pro-migration effect of FOXO6 Given the evident significance of Rac1 and FOXO6 in HCC cell migration, coupled with the confirmed direct regulatory influence of FOXO6 on Rac1, we formulated the hypothesis that Rac1 might serve as a mediator of the pro-migratory effects induced by FOXO6. Employing FOXO6 siRNAs, we observed a substantial inhibition of HCC cell migration. However, this inhibition was effectively reversed when wild-type Rac1 was overexpressed concurrently with the transfection of FOXO6 siRNAs (Fig. 7A-7C). Conversely, the overexpression of wild-type FOXO6 led to an enhancement of HCC cell migration. Yet, when endogenous Rac1 was knocked down, it notably diminished the level of HCC cell migration triggered by FOXO6 (Fig. 7A-7C). These compelling findings collectively establish that Rac1 operates downstream in the FOXO6-dependent pro-migration signaling cascade. Discussion Rac1 is known to mediate a wide array of biological processes, encompassing metabolism, differentiation, proliferation, apoptosis, and migration, and plays a pivotal role in the initiation and progression of various tumors 1 , 34 , 35 . On the other hand, prior research has established the connection between FOXO6 and processes such as development, memory consolidation, synaptic function, and glucose metabolism 16 , 36 , 37 . Nevertheless, the involvement of FOXO6 in tumorigenesis and progression remains unclear. In our present study, we have unveiled the upregulation of FOXO6 in liver cancer tissues and cell lines, shedding light on the role of FOXO6 and Rac1 in HCC cell migration. Our results unequivocally establish Rac1 as a direct target gene of FOXO6 in HCC cells, positioning Rac1 downstream in the FOXO6-dependent pro-migration signaling cascade. This underscores the potential therapeutic relevance of FOXO6 in the context of liver cancer. The pivotal roles of Rac1 in tumor cell migration, invasion, and metastasis are contingent upon its aberrantly heightened activity 1 , 11 , 35 . Therefore, compounds capable of directly impeding Rac1 activation hold promise as key elements in cancer inhibition. Currently, numerous selective Rac1 inhibitors have been developed, including EHop-016, MBQ-167, R-ketorolac, NSC23766, EHT1864, GYS32661, 1D-142, and others, all designed to target Rac1 directly, thereby impairing cancer cell growth, migration, and viability. Certain compounds have even undergone preclinical experimentation, exhibiting favorable inhibitory effects on tumor migration and growth 1 , 35 . However, a substantial challenge persists in confining the effects of these compounds exclusively to cancer cells and tumors, as Rac1 protein is ubiquitously expressed across various tissues. For example, Rac1 plays a pivotal role in axon growth, guidance, and neuronal survival in both the central and peripheral nervous systems. The loss of Rac1 activity in neurons leads to alterations in dendritic spine size and density, thereby impairing traditional measurements of plasticity such as long-term potentiation in the hippocampus 38 . Consequently, this impacts contextual learning and memory 39 . Rac1 also promotes the proliferation, vitality, and cytoskeletal integrity of bladder smooth muscle cells 40 , thereby exerting a potent influence on the contraction of human detrusor smooth muscle 41 . Notably, the Rac1 inhibitors NSC23766 and EHT1864, at a concentration of 100 μM, can significantly hinder platelet diffusion and activation, potentially inducing off-target effects 42 . Furthermore, Rac1 serves as a critical effector in the maintenance of intestinal barrier integrity under normal physiological conditions and during tissue repair processes 43 . Given its myriad roles in physiological activities, directly targeting Rac1 for cancer treatment presents considerable challenges. Interestingly, oncogenic factors frequently promote tumor initiation and development by directly or indirectly upregulating Rac1 activity or expression. Conversely, downregulating Rac1 activity or expression can mitigate the malignancy of tumors. Therefore, identifying factors that specifically induce the upregulation of Rac1 activity presents a promising avenue for drug target exploration. In our investigation, we have discerned that the upregulation of Rac1 in HCC cells is not mediated by HIF-1α, Smad7, miR-142-3p, or miR-137. It is noteworthy that Smad7, miR-142-3p, and miR-137 often function as tumor suppressors in various cancer types. For instance, Smad7 has been reported to suppress HCC cell growth and colony formation by inhibiting the G1-S phase transition 44 . Tumor cell-derived Smad7 has also been shown to inhibit melanoma lung metastasis 45 . Moreover, miR-142-3p has been documented to inhibit lung adenocarcinoma cell proliferation, migration, and invasion, while enhancing apoptosis through the inhibition of NR2F6 46 . Similarly, overexpressing miR-142-3p in cell lines attenuated colorectal cancer cell invasion and migration through the regulation of PKM2-mediated aerobic glycolysis 47 . Furthermore, miR-137 has been found to repress migration and cell motility by targeting COX-2 in non-small cell lung cancer 48 , as well as to suppress proliferation, migration, and invasion of colon cancer cell lines by targeting TCF4 49 . In stark contrast, HIF-1α has been identified as a poor prognostic factor for HCC patients with cirrhosis 50 and has been shown to promote HCC cell migration in an IL-8-NF-κB-dependent manner 51 . A tantalizing question that arises here is whether HIF-1α serves as a downstream mediator in effecting the pro-migratory action of FOXO6-Rac1. This intriguing query is currently the subject of ongoing investigation in our laboratory. Comparatively, FOXO6 was found to be highly expressed in HCC tissues and cells in contrast to adjacent normal liver tissues and normal hepatocytes, signifying its considerable significance in HCC. Knockdown of FOXO6 effectively impeded the migration of HCC cells. Moreover, our analysis of recurrence-free survival unequivocally establishes a significant correlation between elevated FOXO6 expression and an unfavorable prognosis among HCC patients following hepatectomy. Furthermore, our study has compellingly demonstrated that FOXO6 exerts direct transactivation on the Rac1 gene. Firstly, we have demonstrated that the suppression of FOXO6 activity or the knockdown of FOXO6 impairs Rac1 induction, whereas the overexpression of FOXO6 results in elevated Rac1 transcription. Secondly, our ChIP assays have revealed the direct binding of FOXO6 to a FOXO-recognition site in the Rac1 promoter, both in HCC tissues and cell lines. In summation, our research firmly establishes FOXO6 as the specific factor mediating the upregulation of Rac1 total activity in liver cancer. In physiological conditions among adults, the expression of the FOXO6 gene is predominantly localized in the endopiriform nuclei and hippocampus of the brain 16 . Notably, the protein levels of other FOXO isoforms, namely FOXO1, FOXO3, and FOXO4, exhibit no discernible increase in various brain regions among FOXO6 null mice, suggesting a lack of compensation by these isoforms in the absence of FOXO6. Mutant mice lacking FOXO6 display normal thigmotaxis and rearing behavior. Moreover, their motor coordination, interlimb coordination, and neuromuscular capacity remain unaffected. Intriguingly, FOXO6-null mice exhibit unimpaired learning ability, with deficits primarily observed in fear memory consolidation and new learning tasks 36 . These observations underscore the dispensability of FOXO6 for overall survival, brain development, and motor coordination. Our findings, alongside those of previous researchers, underscore the potential of FOXO6 as a highly specific target in tumor However, specific inhibitors capable of blocking FOXO6 transcriptional activity have yet to be developed. Therefore, the design and screening of compounds specifically targeting FOXO6 transcriptional activity, along with their assessment in both physiological and pathological contexts, represent crucial and meaningful avenues for future research. Declarations ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (Major Program) (Grant No.: 82293655), the Provincial Science and Technology Plan Projects in Guangdong Province (Grant No.: 2022A0505050065). AUTHOR CONTRIBUTIONS JFD, BX, JMH and YG conceptualized the project and designed all experiments. JFD, BX, JMH and YG conducted most experiments and wrote the manuscript. QY, SJF and SF performed parts of experiments or provided technical support. JFD and BX analyzed data and organized Fig.s. QY, SJF and SF provided histopathology input and reviewed reports and tumor samples. JFD and BX did the statistical analyzes and data interpretation. JFD, BX, JMH and YG supervised the project and contributed to revise the manuscript. All authors reviewed the final version. CONFLICT OF INTEREST The authors have no potential conflicts of interest to disclose. DATA AVAILABILITY STATEMENT The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. References Ma, N., Xu, E., Luo, Q. & Song, G. Rac1: A Regulator of Cell Migration and a Potential Target for Cancer Therapy. Molecules 28 , doi:10.3390/molecules28072976 (2023). Wu, L. et al. MicroRNA-142-3p, a new regulator of RAC1, suppresses the migration and invasion of hepatocellular carcinoma cells. FEBS letters 585 , 1322-1330, doi:10.1016/j.febslet.2011.03.067 (2011). Zhou, H. C. et al. Dual and opposing roles of the androgen receptor in VETC-dependent and invasion-dependent metastasis of hepatocellular carcinoma. Journal of hepatology 75 , 900-911, doi:10.1016/j.jhep.2021.04.053 (2021). Lo, L. H., Lam, C. Y., To, J. C., Chiu, C. H. & Keng, V. W. Sleeping Beauty insertional mutagenesis screen identifies the pro-metastatic roles of CNPY2 and ACTN2 in hepatocellular carcinoma tumor progression. 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Translational cancer research 11 , 3803-3813, doi:10.21037/tcr-22-2177 (2022). Bi, W. P., Xia, M. & Wang, X. J. miR-137 suppresses proliferation, migration and invasion of colon cancer cell lines by targeting TCF4. Oncology letters 15 , 8744-8748, doi:10.3892/ol.2018.8364 (2018). Wang, D., Zhang, X., Lu, Y., Wang, X. & Zhu, L. Hypoxia inducible factor 1alpha in hepatocellular carcinoma with cirrhosis: Association with prognosis. Pathology, research and practice 214 , 1987-1992, doi:10.1016/j.prp.2018.09.007 (2018). Feng, W. et al. HIF-1alpha promotes the migration and invasion of hepatocellular carcinoma cells via the IL-8-NF-kappaB axis. Cellular & molecular biology letters 23 , 26, doi:10.1186/s11658-018-0077-1 (2018). Table 1 Table 1. Correlation between FOXO expression and clinicopathological features in 37 patients with the recurrence of HCC after hepatectomy Characteristics FOXO positive (n/N) (%) P value TNM stage 0.189 I-II 7/13 (54) III-IVa 18/24 (75) Tumor diameter 0.923 < 5 cm 15/22 (68) ≥ 5 cm 10/15 (67) Tumor number 0.523 20 μg/L 19/25 (76) Histological differentiation 0.04* Good 3/8 (38) Moderate and poor 22/29 (76) χ2 test; TNM: Tumor, node and metastasis; AFP: α fetoprotein; *, P <0.05 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3782217","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":267686808,"identity":"801eb254-f632-408b-af2c-ed18bcf17da3","order_by":0,"name":"Jingfang Diao","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jingfang","middleName":"","lastName":"Diao","suffix":""},{"id":267686809,"identity":"b73695cb-2784-47da-995f-4d786d02e688","order_by":1,"name":"Bo Xie","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYDACZgY2BsY/NkAGcwNMzIAILQ1pQAYjsVoYwFoOA2litRgcZz724OOO89H87YwNjD/b6hIb2Ju3STDU3MGpRbKZLd1w5pnbuTMOMzYw87YdTmzgOVYmwXDsGU4t/Mw8ZtI8bLdzG0BaGNsOJDZI5JhJQJyKwyPM/N+AWs7lzj8Mc5j8G/xagLawSfO2HcjdANTCwNvGDLSFB78WoF/MDWecSc7dCNRymOfcYeM2nrRii4RjuLUYnD/87MGHCrvceecPH3z4o6xOtp/98MYbH2pwa0EBBxjZQNEEBAnEaQCBP8QrHQWjYBSMgpEDAPVYU3+sHMaRAAAAAElFTkSuQmCC","orcid":"","institution":"Sun Yat-sen University","correspondingAuthor":true,"prefix":"","firstName":"Bo","middleName":"","lastName":"Xie","suffix":""},{"id":267686810,"identity":"fd99b183-4c4d-4e8c-9f67-a81819e1e375","order_by":2,"name":"Qing Ye","email":"","orcid":"","institution":"Guangdong Province Traditional Chinese Medical Hospital, Guangzhou University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Ye","suffix":""},{"id":267686811,"identity":"684c65b1-7a7e-4d40-a738-f391dcd355c8","order_by":3,"name":"Shunjun Fu","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shunjun","middleName":"","lastName":"Fu","suffix":""},{"id":267686812,"identity":"e7c71fe3-884b-4d7d-b6eb-6cddc59c6d89","order_by":4,"name":"Xuewen Liu","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xuewen","middleName":"","lastName":"Liu","suffix":""},{"id":267686813,"identity":"fbcbb6ce-8d10-4bb0-8159-e0e957764d82","order_by":5,"name":"Junming He","email":"","orcid":"","institution":"Guangdong Province Traditional Chinese Medical Hospital, Guangzhou University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Junming","middleName":"","lastName":"He","suffix":""},{"id":267686814,"identity":"d5476651-756a-49e0-a286-50d8b73bd972","order_by":6,"name":"Yi Gao","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Gao","suffix":""}],"badges":[],"createdAt":"2023-12-20 14:14:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3782217/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3782217/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49796405,"identity":"0b8ec207-70e4-4e57-977a-8304d47ff38d","added_by":"auto","created_at":"2024-01-18 07:25:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2310658,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe upregulation of Rac1 expression contributes to HCC cell migration. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eB\u003c/strong\u003e) Total RNA or total protein of human normal hepatic cell lines (THLE-2 and THLE-3) and human HCC cell lines (SNU-182, SNU-475 and HepG2) were extracted and analyzed by RT-PCR or Western blotting. (\u003cstrong\u003eC\u003c/strong\u003e) Tissue sections obtained from HCC patients were immunostained for Rac1 (Green) and DAPI (Blue). (\u003cstrong\u003eD\u003c/strong\u003e) SNU-182, SNU-475 and HepG2 cells invasion and migration after 24 h Rac1 siRNA transfection were examined by transwell chamber assays. (\u003cstrong\u003eE\u003c/strong\u003e and \u003cstrong\u003eF\u003c/strong\u003e) Total protein of human normal hepatic cell lines (THLE-2 and THLE-3) and human HCC cell lines (SNU-182, SNU-475 and HepG2) were extracted, then analyzed the total activity of Rac1 (\u003cstrong\u003eE\u003c/strong\u003e) or analyzed the activity of Rac1 according to the total Rac1 protein (\u003cstrong\u003eF\u003c/strong\u003e). (\u003cstrong\u003eG\u003c/strong\u003e) THLE-2 and SNU-475 cells were transfected with or without Rac1 siRNA for 48 h, then total protein was extracted and analyzed the total activity of Rac1. Adjacent normal liver tissues, ANL tissue; Rac1 siRNA, si\u003cem\u003erac1\u003c/em\u003e. All data in this Fig. represent the means±SEM of three independent experiments. *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3782217/v1/72d5f4337cb292e8e83fa34a.png"},{"id":49796404,"identity":"a4d21359-beb0-45ba-88de-172f987703b2","added_by":"auto","created_at":"2024-01-18 07:25:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1786553,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression upregulation of Rac1 is not mediated by HIF-1α, Smad7, miR-142-3p or miR-137. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Human normal hepatic cell lines (THLE-2 and THLE-3) and human HCC cell lines (SNU-182, SNU-475 and HepG2) were transfected with or without Rac1 gene promoter (nucleotides +2825 to -584) based luciferase reporter (Rac1-LucR) for 12h, then dual-luciferase reporter assays were performed. (\u003cstrong\u003eB\u003c/strong\u003e and\u003cstrong\u003e C\u003c/strong\u003e) Total protein form HCC patients, Human normal hepatic cell lines (THLE-2 and THLE-3) and human HCC cell lines (SNU-182, SNU-475 and HepG2) were extracted and analyzed by Western blotting. (\u003cstrong\u003eD\u003c/strong\u003e) Tissue sections obtained from HCC patients were immunostained for Ser-249 phosphorylated Smad7 (Green) and DAPI (Blue). (\u003cstrong\u003eE \u003c/strong\u003eand\u003cstrong\u003e F\u003c/strong\u003e) SNU-475 cells were transfected with or without HIF-1α, Smad7, miR-142-3p or miR-137 siRNA for 48 h, followed by transfecting with Rac1-LucR for 12 h, then cell proteins were extracted and analyzed by Western blotting (\u003cstrong\u003eE\u003c/strong\u003e) or dual-luciferase reporter assays were performed (\u003cstrong\u003eF\u003c/strong\u003e). (\u003cstrong\u003eG\u003c/strong\u003e) SNU-475 cells were transfected with or without HIF-1α, Smad7, miR-142-3p or miR-137 siRNA for 48h, then total RNA was extracted and analyzed the total mRNA of Rac1. All data in this Fig. represent the means±SEM of three independent experiments. *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3782217/v1/2f44b4bfb476b3153e79b975.png"},{"id":49796407,"identity":"6c5cea03-1a0d-45b1-bbf9-a51f48e63787","added_by":"auto","created_at":"2024-01-18 07:25:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":369384,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe FOXO binding site is required for Rac1 promoter activation. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Human Rac1 promoter 5′ sequential deletion constructs. Fragments of different lengths of the Rac1 promoter with the same 3′ end were cloned into pGL3-Basic. THLE-2 and SNU-475 cells were transfected with each of the Rac1 promoter-based reporters for 12 h, then dual-luciferase reporter assays were performed. (\u003cstrong\u003eB\u003c/strong\u003e) Promoter analysis has identified a typical Egr-1 binding site between nucleotides -31/-23 (5′-CGCCGCCGC-3′) and a potential FOXO binding site between nucleotides -208/-202 (5′-TTTCC\u003cu\u003eTGTTTCT\u003c/u\u003eCTGC-3′) in the core region of the Rac1 promoter. (\u003cstrong\u003eC\u003c/strong\u003e) Mutations within the potential Egr-1 or FOXO binding site. (\u003cstrong\u003eD\u003c/strong\u003e) THLE-2 and SNU-475 cells were transfected with each of the Rac1-LucR, Rac1-LucR-MtEgr-1 or Rac1-LucR-MtFOXO for 12 h, then dual-luciferase reporter assays were performed. (\u003cstrong\u003eE\u003c/strong\u003e) Indicated cells were transfected with or without Egr-1 siRNAs for 12 h, followed by transfecting with Rac1-LucR for 12 h, then dual-luciferase reporter assays were performed. All data in this Fig. represent the means±SEM of three independent experiments. *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3782217/v1/175454c489e3cfa832df4657.png"},{"id":49796410,"identity":"b663dd98-d70e-4bd3-bf8c-c2da42532b4e","added_by":"auto","created_at":"2024-01-18 07:25:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":789804,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFOXO6 mediates the expression of Rac1. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Total protein form HCC patients, Human normal hepatic cell lines (THLE-2 and THLE-3) and human HCC cell lines (SNU-182, SNU-475 and HepG2) were extracted and analyzed by Western blotting. (\u003cstrong\u003eB\u003c/strong\u003e) The nuclear protein or cytoplasm protein were extracted from HCC patients, THLE-2 cells or SNU-475 cells and analyzed by Western blotting. (\u003cstrong\u003eC\u003c/strong\u003e) SNU-475 cells were transfected with or without indicated siRNAs for 12 h, followed by transfecting with Rac1-LucR for 12 h, then dual-luciferase reporter assays were performed. (\u003cstrong\u003eD\u003c/strong\u003e) SNU-475 cells were transfected with or without indicated siRNAs for 12 h, then total RNA was extracted and analyzed the total mRNA of Rac1. (\u003cstrong\u003eE\u003c/strong\u003e) SNU-475 cells were transfected with or without indicated siRNAs for 12 h, then total protein was extracted for Western blotting analysis. Adjacent normal liver tissues, ANLT; HCC tissues, HT; Nuclear protein, Nu; cytoplasm protein, Cyto. All data in this Fig. represent the means±SEM of three independent experiments. *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3782217/v1/bf9e819cde88018a9bbfae7c.png"},{"id":49796409,"identity":"73843b19-dbda-4e0f-8ab9-2f61aad3671d","added_by":"auto","created_at":"2024-01-18 07:25:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":815062,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFOXO6 is transcriptionally activated and associates with the early recurrence of HCC after hepatectomy. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e \u003cstrong\u003eupper panel\u003c/strong\u003e) Total protein form HCC patients were extracted and analyzed by Western blotting. (\u003cstrong\u003eA\u003c/strong\u003e \u003cstrong\u003elower panel\u003c/strong\u003e) Tissue sections obtained from HCC patients were immunostained for FOXO6. (\u003cstrong\u003eB\u003c/strong\u003e) Total protein form Human normal hepatic cell lines (THLE-2 and THLE-3) and human HCC cell lines (SNU-182, SNU-475 and HepG2) were extracted and analyzed by Western blotting. (\u003cstrong\u003eC\u003c/strong\u003e) A retrospective analysis of FOXO6 expression was evaluated in relation to clinicopathological features in 37 patients with the recurrence of HCC after hepatectomy. Adjacent normal liver tissues, ANLT; HCC tissues, HT. All data in this Fig. represent the means±SEM of three independent experiments. *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3782217/v1/1acfd04d5cbcfad9481e8dae.png"},{"id":49797039,"identity":"18404cc4-2303-43b0-8000-19710dc552f9","added_by":"auto","created_at":"2024-01-18 07:33:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":514391,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFOXO6 is transcriptionally activated and directly activates Rac1 gene. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Cell lysates from SNU-475 cells or HCC tissues were subjected to Immunoprecipitation (IP) analysis using the Phosphor-Ser184-FOXO6 antibody for two times, then Western blotting analysis with the indicated antibodies demonstrates the IP specificity and efficiency. (B and C) Cell lysates from indicated cells and tissues were subjected to ChIP analysis using the FOXO6 antibody. (\u003cstrong\u003eB\u003c/strong\u003e) Western blotting analysis with the FOXO6 antibody demonstrates the immunoprecipitation (IP) specificity and efficiency. (\u003cstrong\u003eC\u003c/strong\u003e) DNA isolated and purified from immunoprecipitated material was amplified by PCR (upper panel) or Q-PCR (lower panel) with primers spanning the FOXO binding site (Primer: Fs/Rs) or primers far away from the FOXO binding site (Primer: Ff/Rf) of the Rac1 gene promoter. All data in this Fig. represent the means±SEM of three independent experiments. *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3782217/v1/539fbbc8bb4cb0d3ee7a29cd.png"},{"id":49796408,"identity":"9c850ccb-06f9-4b4d-be4f-8881adeb8c9a","added_by":"auto","created_at":"2024-01-18 07:25:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":989758,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRac1 acts downstream to mediate the pro-migration effect of FOXO6. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) SNU-182 cells invasion and migration after 24 h FOXO6 siRNA, Rac1 siRNA, FOXO6, Rac1, FOXO6 siRNA+Rac1 or Rac1 siRNA+FOXO6 transfection were examined by transwell chamber assays (\u003cstrong\u003eA left and middle panel\u003c/strong\u003e), the number of invading tumor cells was counted using 5 high-intensity fields (\u003cstrong\u003eA right panel\u003c/strong\u003e). (\u003cstrong\u003eB\u003c/strong\u003e) SNU-475 cells or (\u003cstrong\u003eC\u003c/strong\u003e) HepG2 cells invasion and migration after 24 h FOXO6 siRNA, Rac1 siRNA, FOXO6, Rac1, FOXO6 siRNA+Rac1 or Rac1 siRNA+FOXO6 transfection were examined by transwell chamber assays, the number of invading tumor cells was counted using 5 high-intensity fields. All data in this Fig. represent the means±SEM of three independent experiments. *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3782217/v1/60a5c02f93d064a95d9a616c.png"},{"id":61795153,"identity":"21932ecd-610f-49d0-b6db-ae68ad49ff5b","added_by":"auto","created_at":"2024-08-05 16:19:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9267633,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3782217/v1/ed40e6a6-325c-4cd2-b298-2cdae0e18fb0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"FOXO6 specifically mediates overactivation of Rac1 in hepatocellular carcinoma","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eRac1 has long been recognized for its pivotal role in the development and metastasis of liver cancer. Notably, the Rac1 protein exhibits heightened expression in invasive hepatocellular carcinoma (HCC) cell lines, and its functionality is closely associated with cell motility and the aggregation of cytoskeletal components \u003csup\u003e1\u003c/sup\u003e. Inhibiting Rac1 activity has been demonstrated to trigger microfilament depolymerization, consequently diminishing cell motility \u003csup\u003e2,3\u003c/sup\u003e. Importantly, Rac1 has been identified as a key player in the growth of primary HCC tumors and the progression of HCC metastasis \u003csup\u003e4\u003c/sup\u003e. As of now, Rac1 is regarded as a promising target for curtailing the acquired and intrinsic resistance of liver tumors and their metastatic potential \u003csup\u003e5\u003c/sup\u003e. Furthermore, it is worth noting that Rac1 overexpression has been linked to the development and advancement of gastric cancer, testicular cancer, and breast cancer \u003csup\u003e6\u0026ndash;8\u003c/sup\u003e. Nevertheless, it is imperative to acknowledge that Rac1 is ubiquitously expressed across various tissues, and any disruption in Rac1 signaling may yield deleterious effects. This is primarily due to the involvement of Rac1-driven cellular processes in numerous physiological and pathological conditions. These encompass actin cytoskeletal reorganization, cell transformation, induction of DNA synthesis, superoxide production, axonal guidance, cell migration, neuronal polarization, cancer, cardiovascular disease, neurodegenerative disease, pathological inflammatory responses, kidney disease, and infectious disease \u003csup\u003e1,9\u0026ndash;11\u003c/sup\u003e. Hence, it may be prudent to prioritize the identification of factors specifically responsible for elevating Rac1 activity as potential drug targets, as opposed to directly inhibiting Rac1 activity itself.\u003c/p\u003e \u003cp\u003eForkhead box protein O6 (FOXO6), a member of the forkhead family, has remained relatively enigmatic with regard to its involvement in tumorigenesis. Nonetheless, recent investigations have shed some light on its relevance. FOXO6 expression has been observed in HCC tissues, correlating with oxidative stress levels \u003csup\u003e12\u003c/sup\u003e. Additionally, overexpression of FOXO6 has been found to promote tumorigenicity in gastric cancer cells \u003csup\u003e13\u003c/sup\u003e, and it is highly upregulated in both breast cancer and colorectal cancer tissues \u003csup\u003e14,15\u003c/sup\u003e. In adult physiology, FOXO6 gene expression is primarily confined to the endopiriform nuclei and hippocampus of the brain \u003csup\u003e16\u003c/sup\u003e. Nonetheless, the precise mechanisms underlying FOXO6's role in carcinogenesis and progression remain poorly understood.\u003c/p\u003e \u003cp\u003eIn the present study, we have substantiated the up-regulation of both FOXO6 and Rac1 expression in HCC tissues and elucidated their involvement in HCC cell migration. Our findings unequivocally establish Rac1 as a direct target gene of FOXO6 in HCC cells, positioning Rac1 as a downstream component of the FOXO6-dependent pro-migration signaling cascade. These revelations underscore the potential of FOXO6 as a therapeutic target for patients grappling with liver cancer.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eCells were cultured as described in our previous study \u003csup\u003e17,18\u003c/sup\u003e. Human normal hepatic cell lines (THLE-2 and THLE-3) and human HCC cell lines (SNU-182, SNU-475 and HepG2) were all purchased from American Type Culture Collection (ATCC). All cell lines used, were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 100 U/ml penicillin-streptomycin (Gibco) in a 5% CO2 humidified incubator at 37\u0026deg;C. HUVEC was cultured in LSGS-supplemented Medium 200 (Gibco) with 10% FBS and 100 U/ml penicillin-streptomycin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePatient tissue specimens\u003c/h2\u003e \u003cp\u003eThe samples were collected between 2015 and 2023 at Guangdong Provincial Hospital of Traditional Chinese Medicine (Guangzhou, China). The HCC cases selected were based on a clear pathological diagnosis after surgery and follow-up data and had not received neoadjuvant chemotherapy or radiotherapy. HCC tissues and matched tumor-adjacent morphologically normal liver epithelial tissues were frozen and stored in liquid nitrogen. The collected HCC tissues and adjacent normal liver tissues used in this study were in accordance with the ethical standards of the institute research ethics committee of Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou University of Chinese Medicine (Reference No. ZE2023-304-01) and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all participants before the procedure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical chemistry (IHC) analyzes\u003c/h2\u003e \u003cp\u003eTissues were cut into 5-\u0026micro;m sections and processed for IHC using a standard two-step technique as demonstrated previously. Alkaline phosphatase substrate kit III was obtained from Vector Laboratories, Inc. Anti-mouse alkaline phosphatase conjugate (Sigma) was applied to each section for 1 h at room temperature, sections were washed, and alkaline phosphatase substrate was applied for 20 min. Before counterstaining and dehydration, the sections were then subjected to IHC for different antibodies as the chromagen. Sections were washed and counterstained with Mayer\u0026rsquo;s hemalum. Microscopy was performed using an Olympus Fluoview confocal microscope or an Olympus Provis microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence analyzes\u003c/h2\u003e \u003cp\u003eTissues were cut into 5 \u0026micro;m sections and processed for immunofluorescence using a standard two-step technique. Tissue sections were washed twice with PBS and followed by permeabilization with 0.1% Triton X-100 in TBS and blocking in 3% donkey serum. Tissue sections were then washed twice in PBS and treated by cold blocking buffer for 1h. After sequential treatment with NH\u003csub\u003e4\u003c/sub\u003eCl (50 mM in 20 mM glycine) for 10 min, the indicated antibody (1:100 in bovine serum albumin) was added and incubated overnight at 4\u0026deg;C. After an additional incubation for 1 h at room temperature with Hoechst 33258 and Alexa Fluor\u0026reg; 488 -conjugated secondary antibody (Abcam) (1:400 in bovine serum albumin), the slides were mounted in anti-fading solution (Permafluor, Beckman Coulter, Krefeld, Germany) and stored at 4\u0026deg;C, followed by confocal laser-scanning microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTranswell migration assay\u003c/h2\u003e \u003cp\u003eCells were plated at 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well in 8.0-mm pore-sized 24-well Transwell inserts (Corning) with serum-free RPMI-1640, and the lower chamber was filled with 0.6 ml/well of RPMI-1640 supplemented with 2% FBS. The cells were incubated at 37\u0026deg;C for 24h. Thelower side of the filter was fixed in 4% paraformaldehyde (Electron Microscopy Science) and stained with 0.5% Crystal violet (Sigma-Aldrich) for 20 min, followed by examination under a microscope whereby 5 randomly non-overlapping high-power fields (HPFs, \u0026times;200) were selected to count the stained migrated cells. All the experiments in each group were performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRac1 activity assay\u003c/h2\u003e \u003cp\u003eThe activity of Rac1 (GTP bound form) was assayed in the ovarian protein extract using G-LISA Rac1 activation assay Biochem Kit, as per the manufacturer\u0026rsquo;s instructions and that is already validated. Briefly, a total of 50 \u0026micro;g protein extract was added to each corresponding well pre-coated with Rac-GTP-binding protein. This was incubated at 4\u0026deg;C for 30 min followed by successive incubation with 50 \u0026micro;l of anti-Rac1 for 45 min. Later, secondary antibody conjugated with HRP (50 \u0026micro;l) was incubated for 45 minutes. Subsequently, 50 \u0026micro;l of HRP detection reagent was added to each well, followed by incubation for another 20 min. The reaction was stopped by the addition of 50 \u0026micro;l of HRP stop solution and the absorbance was recorded at 490 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eReal-Time Polymerase Chain Reaction (Real-time PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using the EastepTM Universal RNA Extraction Kit (Promega, Madison, WI) and transversely transcribed with the GoScriptTM Reverse Transcription System (Promega, Madison, WI) according to the manufacturer\u0026rsquo;s instruction. Real-time PCR was performed in triplicate with GoTaq\u0026reg; qPCR Master Mix (Promega, Madison, WI) and run on the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Framingham, MA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eWestern blotting analysis was performed as described in our previous study\u003csup\u003e17,18\u003c/sup\u003e. Briefly, the protein extracts from cells or tissues were separated by 8% or 10% SDS‒PAGE, transferred to a PVDF membrane and incubated with the indicated primary antibody. The signal was detected by an ECL detection system (GE Healthcare).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003esiRNA interference\u003c/h2\u003e \u003cp\u003eRNA interference analysis was performed as described in our previous study. Specific siRNAs were from Dharmacon (ONTARGETplus SMARTpool) and Santacruz; the negative control (NC) siRNA (no silencing small RNA fragment) was synthesized by GenChem Co. (Shanghai, China). siRNAs were transfected into cells using Lipofectamine RNAiMAX (iMAX) (Life Technologies, InvitrogenTM, Cat No: #13778150).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCo-immunoprecipitation\u003c/h2\u003e \u003cp\u003eCo-immunoprecipitation was performed as described previously \u003csup\u003e19\u003c/sup\u003e. For in vivo protein interaction, cell extracts were prepared by solubilizing 10\u003csup\u003e7\u003c/sup\u003e cells in 1 ml of cell lysis buffer made of 1% Triton X-100, 150 mM NaCl, 20 mM Tris-Cl at pH 7.4, 1 mM EDTA, 1 mM EGTA, 1 mM Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e, 2.5 mM pyrophosphate, 1 mM glycerol phosphate and protease inhibitor mixture for 10 min at 4\u0026deg;C. After brief sonication, the lysates were cleared by centrifugation at 15,000 \u0026times; g for 10 min at 4\u0026deg;C, and the cell extract was immunoprecipitated with 2 \u0026micro;g of antibodies and incubated with 100 \u0026micro;l of protein G plus protein A-agarose for 12 h at 4\u0026deg;C by continuous inversion. Immunocomplexes were pelleted, washed 3 times, boiled in Laemmli buffer and analyzed by Western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDual-luciferase reporter assays\u003c/h2\u003e \u003cp\u003eA dual-luciferase reporter assay was performed as described in our previous study \u003csup\u003e20\u003c/sup\u003e. Constructs were transfected into cells using Lipofectamine 2000. For the dual-luciferase reporter assays, cells were transfected with 1 \u0026micro;g of a luciferase reporter plasmid and 200 ng of the pRL-CMV Renilla luciferase reporter plasmid (Promega). After transfection, cells were kept in conditioned media for 12 or 24 h and then transferred to treatment media for 12 h. Firefly luciferase activity was normalized to Renilla luciferase activity according to the protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eChromatin immunoprecipitation assays (ChIP)\u003c/h2\u003e \u003cp\u003eChIP was performed as described in our previous studies \u003csup\u003e20\u003c/sup\u003e. Briefly, ChIP assays were performed using the ChIP assay kit (Upstate Cell Signaling Solutions) according to the manufacturer\u0026rsquo;s instructions. Egr-1 antibody (#4153, Cell Signaling Technology) was used for immunoprecipitation. Purified DNA was subjected to PCR amplification using primers spanning the Rac1 gene promoter FOXO site.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyzes were performed using PASW Statistics 22.0 (SPSS Inc., Chicago, IL), with the exception of the significance in bar graphs, in which case analyzes were performed by applying the independent t test using Microsoft Office Excel software (Microsoft Corp., Redmond, WA). The statistics in the graphs represent the means\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.s. Bars represent at least three independent experiments. A p value of less than 0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e\u003cp\u003e\u003cstrong\u003eEthical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study were approved by ethical standards of the institute research ethics committee of Guangdong Provincial Hospital of Traditional Chinese Medicine，Guangzhou University of Chinese Medicine (Reference No. ZE2023-304-01) and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all participants before the procedure.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eUpregulation of Rac1 expression contributes to HCC cell migration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt has been established that Rac1 can facilitate the migration of HCC cells. In our study, we observed a significant elevation in Rac1 mRNA and protein expression levels in human HCC cell lines compared to human normal hepatic cell (NHC) lines (Fig. 1A and 1B). Additionally, we confirmed a marked upregulation of Rac1 in HCC tissues in comparison to adjacent normal liver tissues (Fig. 1C). To substantiate the role of Rac1 in promoting HCC cell migration, we employed specific siRNAs to partially inhibit Rac1 upregulation, and our findings confirmed the significance of Rac1 in this process (Fig. 1D).\u003c/p\u003e\n\u003cp\u003eTo assess whether Rac1 activity was indeed heightened in HCC cells, we conducted a comparative analysis of total Rac1 activity and relative Rac1 activity between HCC cells and NHC cells. Interestingly, we observed an increase in the total activity of Rac1 in HCC cells (Fig. 1E). However, the relative activities of Rac1 remained unaltered (Fig. 1F). Furthermore, the inhibition of Rac1 expression prevented the escalation of total Rac1 activity without affecting the relative activity of Rac1 (Fig. 1G). These findings collectively demonstrate that the upregulation of Rac1 expression contributes to the activation of Rac1 and, consequently, the migration of HCC cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression upregulation of Rac1 is not mediated by HIF-1\u0026alpha;, Smad7, miR-142-3p or miR-137\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn HCC, previous reports have indicated an elevation in Rac1 expression \u003csup\u003e2\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e21\u003c/sup\u003e. To substantiate the transcriptional upregulation of the Rac1 gene, we transfected cells with a luciferase reporter construct containing the Rac1 gene promoter sequence (nucleotides +2825 to -584), referred to as Rac1-LucR. Our observations revealed an approximately fourfold increase in Rac1-LucR activity in HCC cells when compared to NHC cells (Fig. 2A), thereby confirming the transcriptional upregulation of Rac1 in HCC cells.\u003c/p\u003e\n\u003cp\u003eSmad7, a pivotal inhibitor of the TGF\u0026beta;-Smad signaling pathway, has the capacity to augment Rac1 promoter activity by attenuating CtBP1 binding to the mouse Rac1 promoter. This effect has been observed in both mouse oral mucositis and human oral keratinocytes \u003csup\u003e22\u003c/sup\u003e. However, our investigation uncovered a downregulation of Smad7 in human HCC tissues and cell lines relative to adjacent normal tissues and NHC lines (Fig. 2B and 2C). Furthermore, the phosphorylation and nuclear localization of Ser-249-Smad7, which influence the transcriptional activity of Smad7 \u003csup\u003e23\u003c/sup\u003e, exhibited a decrease in human HCC tissues compared to adjacent normal tissues (Fig. 2D). Notably, the use of Smad7 siRNAs did not exert any influence on the promoter activity, mRNA levels, or protein levels of Rac1 (Fig. 2E-2G), thus confirming that Smad7 does not function as a regulator of Rac1 expression in HCC cells.\u003c/p\u003e\n\u003cp\u003ePrevious studies have proposed regulatory roles for HIF-1\u0026alpha;, miR-142-3p, or miR-137 in modulating Rac1 expression \u003csup\u003e2\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e22\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e24\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e25\u003c/sup\u003e. However, our experiments employing HIF-1\u0026alpha;, miR-142-3p, or miR-137 siRNAs revealed no discernible impact on Rac1 promoter activity, mRNA levels, or protein levels (Fig. 2E-2G). This underscores an HIF-1\u0026alpha;, miR-142-3p, or miR-137-independent mechanism governing Rac1 expression in HCC cells.\u003c/p\u003e\n\u003cp\u003eCollectively, these findings suggest that the upregulation of Rac1 expression in HCC cells is not mediated by HIF-1\u0026alpha;, Smad7, miR-142-3p, or miR-137.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe FOXO binding site is required for Rac1 promoter activation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo ascertain the transcription factor responsible for regulating the activity of the Rac1 gene (Gene ID: 5879) promoter \u003csup\u003e26\u003c/sup\u003e, we conducted an investigation to pinpoint the core sequences essential for promoter activation. We designed a series of reporter constructs featuring deletions of the 5\u0026apos;-flanking region of the Rac1 promoter, all of which retained the same 3\u0026apos; end. Among these, one deletion construct (encompassing nucleotides -231 to +455) exhibited a response analogous to that of the original construct (encompassing nucleotides -1918 to +455) in HCC cells. In contrast, another deletion construct (encompassing nucleotides +16 to +455) effectively abolished the heightened promoter activity (Fig. 3A). Consequently, we delineated the principal Rac1 transcriptional control element to the region spanning nucleotides -231 to +16.\u003c/p\u003e\n\u003cp\u003ePromoter analysis unveiled the presence of a typical Egr-1 binding site situated between nucleotides -31/-23 (5\u0026apos;-CGCCGCCGC-3\u0026apos; or 3\u0026apos;-GCGGCGGCG-5\u0026apos;) in the core region of the Rac1 promoter (Fig. 3B). Notably, this putative Egr-1 site coincided with its classical binding site in the PTEN promoter \u003csup\u003e27\u003c/sup\u003e. To assess the role of this putative Egr-1 site in Rac1 promoter activation, we introduced mutations within the site into the reporter vector. Specifically, we created a human mutant Egr-1 binding site Rac1 reporter gene designated as Rac1-LucR-MtEgr-1 (Fig. 3C). Intriguingly, our results revealed that these mutations did not exert any influence on the activity of the Rac1 promoter in HCC cells (Fig. 3D). Furthermore, our experiments involving Egr-1 siRNAs demonstrated their inability to impact the promoter activity of Rac1 (Fig. 3E), thus confirming that Egr-1 does not serve as a regulator of Rac1 expression in HCC cells.\u003c/p\u003e\n\u003cp\u003eAdditionally, our promoter analysis unveiled a potential binding site for Forkhead-box O subclass proteins (FOXO) between nucleotides -208/-202 (5\u0026apos;-TTTCC\u003cu\u003eTGTTTCT\u003c/u\u003eCTGC-3\u0026apos; or 3\u0026apos;-GCAG\u003cu\u003eAGAAACA\u003c/u\u003eGGAAA-5\u0026apos;) in the core region of the Rac1 promoter (Fig. 3B). Notably, this putative FOXO site contains the core sequence recognized by FOXO proteins \u003csup\u003e28\u003c/sup\u003e. To assess the role of this putative FOXO binding site in Rac1 promoter activation, we introduced mutations within the site into the reporter vector, generating a human mutant FOXO site Rac1 reporter gene referred to as Rac1-LucR-MtFOXO (Fig. 3C). Our results unequivocally demonstrated that these mutations disrupted the activation of the Rac1 promoter in HCC cells (Fig. 3D). These findings conclusively establish the significance of the putative FOXO site (nucleotides -208 to -202) in Rac1 promoter activation in HCC cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFOXO6 mediates the expression of Rac1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u0026quot;O\u0026quot; subclass of the human FOX family encompasses four members (FOXO1, FOXO3, FOXO4, and FOXO6), each contributing to the control of various metabolic processes, cell survival, cellular proliferation, DNA damage repair responses, and stress resistance \u003csup\u003e29\u003c/sup\u003e. As transcription factors, the nuclear localization of FOXO proteins is a fundamental prerequisite for the regulation of their target gene expression. Previous studies conducted in mammalian cell lines have established that when Akt phosphorylates FOXO1, FOXO3, and FOXO4, these proteins relocate from the nucleus to the cytosol \u003csup\u003e30\u003c/sup\u003e. \u003c/p\u003e\n\u003cp\u003eIn our current study, we observed no difference in the phosphorylation of FOXO1 (Thr24), FOXO3 (Thr32), FOXO4 (Ser197), or their upstream kinase Akt (Ser473) when comparing human HCC tissues and cell lines with adjacent normal tissues and NHC lines. This observation held true despite a significant elevation in Rac1 protein levels being evident in human HCC tissues and HCC cell lines (Fig. 4A). Intriguingly, we noted that FOXO1, FOXO3, and FOXO4 predominantly exhibited cytosolic localization in both HCC and NHC cells. In stark contrast, FOXO6 primarily resided in the nucleus of HCC cells when compared to NHC cells (Fig. 4B). To further elucidate which FOXO member is involved in the regulation of Rac1 expression, we employed specific siRNAs targeting FOXO members. Our findings indicated that only siRNAs targeting FOXO6 had a discernible impact on the promoter activity, mRNA levels, and protein levels of Rac1 (Fig. 4C-4E). This underscores a FOXO6-dependent mechanism governing Rac1 expression regulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptional activation of FOXO6 is associated with the early recurrence of HCC after hepatectomy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we embarked on an investigation to assess the correlation between FOXO6 expression and the prognosis of HCC. A prior study had already reported the overexpression of FOXO6 in HCC tissues, thus substantiating its relevance in the pathogenesis of HCC \u003csup\u003e12\u003c/sup\u003e. Our own analysis reaffirmed these findings, as we observed a marked upregulation of FOXO6 protein levels in HCC tissues in comparison to adjacent normal liver tissues (Fig. 5A). Furthermore, the protein levels of FOXO6 were significantly higher in HCC cells compared to NHCs (Fig. 5B). To further elucidate the clinical implications of FOXO6 expression, we conducted a retrospective analysis in 37 patients who experienced HCC recurrence following hepatectomy. This analysis revealed a close association between FOXO6 overexpression and vascular invasion (P=0.007) as well as histological differentiation (P=0.04). However, no significant correlations were established between FOXO6 overexpression and parameters such as tumor, node, metastasis (TNM) stages, tumor diameter, tumor number, and serum AFP levels (Table 1). Subsequent analyses focused on the average duration of HCC recurrence in these 37 patients, which was found to be 17.1 months on average (ranging from 4 to 35 months) post-hepatectomy. Kaplan-Meier survival curves were employed for further assessment, revealing that patients devoid of FOXO6 overexpression experienced significantly longer recurrence-free survival in comparison to those with FOXO6 overexpression (P=0.024, log-rank test) (Fig. 5C). In summary, our recurrence-free survival analysis establishes a significant correlation between increased FOXO6 expression and an adverse prognosis among HCC patients following hepatectomy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptional activation of FOXO6 directly activates Rac1 gene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA previous study has highlighted that FOXO6\u0026apos;s transcriptional activity is subject to regulation not solely based on nuclear localization but also on the phosphorylation status of Ser184 \u003csup\u003e31\u003c/sup\u003e. Specifically, Ser184-phosphorylated FOXO6 is known to interact with 14-3-3\u0026beta;, a scaffold protein, resulting in the sequestration of FOXO6 and its prevention from binding to target promoters \u003csup\u003e32\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e33\u003c/sup\u003e. To investigate whether FOXO6 is indeed activated in HCC tissues and cultured cells, we conducted an immunodepletion study. Our findings indicated that the immunodepletion of Ser184-phosphorylated FOXO6 had only a minor impact on the total FOXO6 levels, with reductions of 16.5% in HCC tissues and 15.8% in SNU-475 cells (Fig. 6A). Thus, it can be inferred that FOXO6 appears to be activated in both HCC tissues and cell lines.\u003c/p\u003e\n\u003cp\u003eIn order to ascertain whether FOXO6 interacts directly with the Rac1 promoter, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, we employed a Chromatin Immunoprecipitation (ChIP) assay. Precipitated immunocomplexes were subjected to Western blot analysis using an anti-FOXO6 antibody. The results revealed the presence of FOXO6 protein cross-linked to DNA in HCC cells and tissues (Fig. 6B). Subsequently, the precipitated DNA was subjected to PCR using primers flanking the FOXO6 binding site in the Rac1 promoter. This assay led to a robust amplification of the DNA fragment from both Rac1-treated cells and mice (Fig. 6C). Thus, our findings affirm that FOXO6 directly binds to the FOX binding site region in the Rac1 promoter, thereby promoting the expression of Rac1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRac1 acts downstream to mediate the pro-migration effect of FOXO6\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the evident significance of Rac1 and FOXO6 in HCC cell migration, coupled with the confirmed direct regulatory influence of FOXO6 on Rac1, we formulated the hypothesis that Rac1 might serve as a mediator of the pro-migratory effects induced by FOXO6. Employing FOXO6 siRNAs, we observed a substantial inhibition of HCC cell migration. However, this inhibition was effectively reversed when wild-type Rac1 was overexpressed concurrently with the transfection of FOXO6 siRNAs (Fig. 7A-7C). Conversely, the overexpression of wild-type FOXO6 led to an enhancement of HCC cell migration. Yet, when endogenous Rac1 was knocked down, it notably diminished the level of HCC cell migration triggered by FOXO6 (Fig. 7A-7C). These compelling findings collectively establish that Rac1 operates downstream in the FOXO6-dependent pro-migration signaling cascade.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eRac1 is known to mediate a wide array of biological processes, encompassing metabolism, differentiation, proliferation, apoptosis, and migration, and plays a pivotal role in the initiation and progression of various tumors \u003csup\u003e1\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e34\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e35\u003c/sup\u003e. On the other hand, prior research has established the connection between FOXO6 and processes such as development, memory consolidation, synaptic function, and glucose metabolism \u003csup\u003e16\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e36\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e37\u003c/sup\u003e. Nevertheless, the involvement of FOXO6 in tumorigenesis and progression remains unclear. In our present study, we have unveiled the upregulation of FOXO6 in liver cancer tissues and cell lines, shedding light on the role of FOXO6 and Rac1 in HCC cell migration. Our results unequivocally establish Rac1 as a direct target gene of FOXO6 in HCC cells, positioning Rac1 downstream in the FOXO6-dependent pro-migration signaling cascade. This underscores the potential therapeutic relevance of FOXO6 in the context of liver cancer.\u003c/p\u003e\n\u003cp\u003eThe pivotal roles of Rac1 in tumor cell migration, invasion, and metastasis are contingent upon its aberrantly heightened activity \u003csup\u003e1\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e11\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e35\u003c/sup\u003e. Therefore, compounds capable of directly impeding Rac1 activation hold promise as key elements in cancer inhibition. Currently, numerous selective Rac1 inhibitors have been developed, including EHop-016, MBQ-167, R-ketorolac, NSC23766, EHT1864, GYS32661, 1D-142, and others, all designed to target Rac1 directly, thereby impairing cancer cell growth, migration, and viability. Certain compounds have even undergone preclinical experimentation, exhibiting favorable inhibitory effects on tumor migration and growth \u003csup\u003e1\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e35\u003c/sup\u003e. However, a substantial challenge persists in confining the effects of these compounds exclusively to cancer cells and tumors, as Rac1 protein is ubiquitously expressed across various tissues. For example, Rac1 plays a pivotal role in axon growth, guidance, and neuronal survival in both the central and peripheral nervous systems. The loss of Rac1 activity in neurons leads to alterations in dendritic spine size and density, thereby impairing traditional measurements of plasticity such as long-term potentiation in the hippocampus \u003csup\u003e38\u003c/sup\u003e. Consequently, this impacts contextual learning and memory \u003csup\u003e39\u003c/sup\u003e. Rac1 also promotes the proliferation, vitality, and cytoskeletal integrity of bladder smooth muscle cells \u003csup\u003e40\u003c/sup\u003e, thereby exerting a potent influence on the contraction of human detrusor smooth muscle \u003csup\u003e41\u003c/sup\u003e. Notably, the Rac1 inhibitors NSC23766 and EHT1864, at a concentration of 100 \u0026mu;M, can significantly hinder platelet diffusion and activation, potentially inducing off-target effects \u003csup\u003e42\u003c/sup\u003e. Furthermore, Rac1 serves as a critical effector in the maintenance of intestinal barrier integrity under normal physiological conditions and during tissue repair processes \u003csup\u003e43\u003c/sup\u003e. Given its myriad roles in physiological activities, directly targeting Rac1 for cancer treatment presents considerable challenges. Interestingly, oncogenic factors frequently promote tumor initiation and development by directly or indirectly upregulating Rac1 activity or expression. Conversely, downregulating Rac1 activity or expression can mitigate the malignancy of tumors. Therefore, identifying factors that specifically induce the upregulation of Rac1 activity presents a promising avenue for drug target exploration. \u003c/p\u003e\n\u003cp\u003eIn our investigation, we have discerned that the upregulation of Rac1 in HCC cells is not mediated by HIF-1\u0026alpha;, Smad7, miR-142-3p, or miR-137. It is noteworthy that Smad7, miR-142-3p, and miR-137 often function as tumor suppressors in various cancer types. For instance, Smad7 has been reported to suppress HCC cell growth and colony formation by inhibiting the G1-S phase transition \u003csup\u003e44\u003c/sup\u003e. Tumor cell-derived Smad7 has also been shown to inhibit melanoma lung metastasis \u003csup\u003e45\u003c/sup\u003e . Moreover, miR-142-3p has been documented to inhibit lung adenocarcinoma cell proliferation, migration, and invasion, while enhancing apoptosis through the inhibition of NR2F6 \u003csup\u003e46\u003c/sup\u003e. Similarly, overexpressing miR-142-3p in cell lines attenuated colorectal cancer cell invasion and migration through the regulation of PKM2-mediated aerobic glycolysis \u003csup\u003e47\u003c/sup\u003e. Furthermore, miR-137 has been found to repress migration and cell motility by targeting COX-2 in non-small cell lung cancer \u003csup\u003e48\u003c/sup\u003e, as well as to suppress proliferation, migration, and invasion of colon cancer cell lines by targeting TCF4 \u003csup\u003e49\u003c/sup\u003e. In stark contrast, HIF-1\u0026alpha; has been identified as a poor prognostic factor for HCC patients with cirrhosis \u003csup\u003e50\u003c/sup\u003e and has been shown to promote HCC cell migration in an IL-8-NF-\u0026kappa;B-dependent manner \u003csup\u003e51\u003c/sup\u003e. A tantalizing question that arises here is whether HIF-1\u0026alpha; serves as a downstream mediator in effecting the pro-migratory action of FOXO6-Rac1. This intriguing query is currently the subject of ongoing investigation in our laboratory.\u003c/p\u003e\n\u003cp\u003eComparatively, FOXO6 was found to be highly expressed in HCC tissues and cells in contrast to adjacent normal liver tissues and normal hepatocytes, signifying its considerable significance in HCC. Knockdown of FOXO6 effectively impeded the migration of HCC cells. Moreover, our analysis of recurrence-free survival unequivocally establishes a significant correlation between elevated FOXO6 expression and an unfavorable prognosis among HCC patients following hepatectomy. Furthermore, our study has compellingly demonstrated that FOXO6 exerts direct transactivation on the Rac1 gene. Firstly, we have demonstrated that the suppression of FOXO6 activity or the knockdown of FOXO6 impairs Rac1 induction, whereas the overexpression of FOXO6 results in elevated Rac1 transcription. Secondly, our ChIP assays have revealed the direct binding of FOXO6 to a FOXO-recognition site in the Rac1 promoter, both in HCC tissues and cell lines. In summation, our research firmly establishes FOXO6 as the specific factor mediating the upregulation of Rac1 total activity in liver cancer.\u003c/p\u003e\n\u003cp\u003eIn physiological conditions among adults, the expression of the FOXO6 gene is predominantly localized in the endopiriform nuclei and hippocampus of the brain \u003csup\u003e16\u003c/sup\u003e. Notably, the protein levels of other FOXO isoforms, namely FOXO1, FOXO3, and FOXO4, exhibit no discernible increase in various brain regions among FOXO6 null mice, suggesting a lack of compensation by these isoforms in the absence of FOXO6. Mutant mice lacking FOXO6 display normal thigmotaxis and rearing behavior. Moreover, their motor coordination, interlimb coordination, and neuromuscular capacity remain unaffected. Intriguingly, FOXO6-null mice exhibit unimpaired learning ability, with deficits primarily observed in fear memory consolidation and new learning tasks \u003csup\u003e36\u003c/sup\u003e. These observations underscore the dispensability of FOXO6 for overall survival, brain development, and motor coordination. \u003c/p\u003e\n\u003cp\u003eOur findings, alongside those of previous researchers, underscore the potential of FOXO6 as a highly specific target in tumor However, specific inhibitors capable of blocking FOXO6 transcriptional activity have yet to be developed. Therefore, the design and screening of compounds specifically targeting FOXO6 transcriptional activity, along with their assessment in both physiological and pathological contexts, represent crucial and meaningful avenues for future research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (Major Program) (Grant No.: 82293655), the Provincial Science and Technology Plan Projects in Guangdong Province (Grant No.: 2022A0505050065).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eJFD, BX, JMH and YG conceptualized the project and designed all experiments. JFD, BX, JMH and YG conducted most experiments and wrote the manuscript. QY, SJF and\u0026nbsp;SF\u0026nbsp;performed parts of experiments or provided technical support. JFD and BX analyzed data\u0026nbsp;and organized Fig.s. QY, SJF and\u0026nbsp;SF provided histopathology input and reviewed reports and tumor samples.\u0026nbsp;JFD and BX\u0026nbsp;did the statistical analyzes and data interpretation.\u0026nbsp;JFD, BX, JMH and YG supervised the project and contributed to revise the manuscript.\u0026nbsp;All authors reviewed the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no potential conflicts of interest to disclose.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMa, N., Xu, E., Luo, Q. \u0026amp; Song, G. 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FoxO6 transcriptional activity is regulated by Thr26 and Ser184, independent of nucleo-cytoplasmic shuttling. \u003cem\u003eThe Biochemical journal\u003c/em\u003e \u003cstrong\u003e391\u003c/strong\u003e, 623-629, doi:10.1042/BJ20050525 (2005).\u003c/li\u003e\n\u003cli\u003eKim, D. H.\u003cem\u003e et al.\u003c/em\u003e FoxO6 integrates insulin signaling with gluconeogenesis in the liver. \u003cem\u003eDiabetes\u003c/em\u003e \u003cstrong\u003e60\u003c/strong\u003e, 2763-2774, doi:10.2337/db11-0548 (2011).\u003c/li\u003e\n\u003cli\u003eLee, S. \u0026amp; Dong, H. H. FoxO integration of insulin signaling with glucose and lipid metabolism. \u003cem\u003eThe Journal of endocrinology\u003c/em\u003e \u003cstrong\u003e233\u003c/strong\u003e, R67-R79, doi:10.1530/JOE-17-0002 (2017).\u003c/li\u003e\n\u003cli\u003ePayapilly, A. \u0026amp; Malliri, A. Compartmentalisation of RAC1 signalling. \u003cem\u003eCurrent opinion in cell biology\u003c/em\u003e \u003cstrong\u003e54\u003c/strong\u003e, 50-56, doi:10.1016/j.ceb.2018.04.009 (2018).\u003c/li\u003e\n\u003cli\u003eBailly, C., Beignet, J., Loirand, G. \u0026amp; Sauzeau, V. Rac1 as a therapeutic anticancer target: Promises and limitations. \u003cem\u003eBiochemical pharmacology\u003c/em\u003e \u003cstrong\u003e203\u003c/strong\u003e, 115180, doi:10.1016/j.bcp.2022.115180 (2022).\u003c/li\u003e\n\u003cli\u003eSalih, D. A.\u003cem\u003e et al.\u003c/em\u003e FoxO6 regulates memory consolidation and synaptic function. \u003cem\u003eGenes \u0026amp; development\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 2780-2801, doi:10.1101/gad.208926.112 (2012).\u003c/li\u003e\n\u003cli\u003eKim, D. H., Zhang, T., Lee, S. \u0026amp; Dong, H. H. FoxO6 in glucose metabolism (FoxO6). \u003cem\u003eJournal of diabetes\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 233-240, doi:10.1111/1753-0407.12027 (2013).\u003c/li\u003e\n\u003cli\u003eHaditsch, U.\u003cem\u003e et al.\u003c/em\u003e A central role for the small GTPase Rac1 in hippocampal plasticity and spatial learning and memory. \u003cem\u003eMolecular and cellular neurosciences\u003c/em\u003e \u003cstrong\u003e41\u003c/strong\u003e, 409-419, doi:10.1016/j.mcn.2009.04.005 (2009).\u003c/li\u003e\n\u003cli\u003eHaditsch, U.\u003cem\u003e et al.\u003c/em\u003e Neuronal Rac1 is required for learning-evoked neurogenesis. \u003cem\u003eThe Journal of neuroscience : the official journal of the Society for Neuroscience\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 12229-12241, doi:10.1523/JNEUROSCI.2939-12.2013 (2013).\u003c/li\u003e\n\u003cli\u003eWang, R.\u003cem\u003e et al.\u003c/em\u003e Rac1 silencing, NSC23766 and EHT1864 reduce growth and actin organization of bladder smooth muscle cells. \u003cem\u003eLife sciences\u003c/em\u003e \u003cstrong\u003e261\u003c/strong\u003e, 118468, doi:10.1016/j.lfs.2020.118468 (2020).\u003c/li\u003e\n\u003cli\u003eLi, B.\u003cem\u003e et al.\u003c/em\u003e Inhibition of Female and Male Human Detrusor Smooth Muscle Contraction by the Rac Inhibitors EHT1864 and NSC23766. \u003cem\u003eFrontiers in pharmacology\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 409, doi:10.3389/fphar.2020.00409 (2020).\u003c/li\u003e\n\u003cli\u003eDutting, S.\u003cem\u003e et al.\u003c/em\u003e Critical off-target effects of the widely used Rac1 inhibitors NSC23766 and EHT1864 in mouse platelets. \u003cem\u003eJournal of thrombosis and haemostasis : JTH\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 827-838, doi:10.1111/jth.12861 (2015).\u003c/li\u003e\n\u003cli\u003eKotelevets, L. \u0026amp; Chastre, E. Rac1 Signaling: From Intestinal Homeostasis to Colorectal Cancer Metastasis. \u003cem\u003eCancers\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, doi:10.3390/cancers12030665 (2020).\u003c/li\u003e\n\u003cli\u003eWang, J.\u003cem\u003e et al.\u003c/em\u003e Inhibitory role of Smad7 in hepatocarcinogenesis in mice and in vitro. \u003cem\u003eThe Journal of pathology\u003c/em\u003e \u003cstrong\u003e230\u003c/strong\u003e, 441-452, doi:10.1002/path.4206 (2013).\u003c/li\u003e\n\u003cli\u003eMa, D., Qiao, L. \u0026amp; Guo, B. Smad7 suppresses melanoma lung metastasis by impairing Tregs migration to the tumor microenvironment. \u003cem\u003eAmerican journal of translational research\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 719-731 (2021).\u003c/li\u003e\n\u003cli\u003eJin, C.\u003cem\u003e et al.\u003c/em\u003e MiR-142-3p suppresses the proliferation, migration and invasion through inhibition of NR2F6 in lung adenocarcinoma. \u003cem\u003eHuman cell\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 437-446, doi:10.1007/s13577-019-00258-0 (2019).\u003c/li\u003e\n\u003cli\u003eRen, J.\u003cem\u003e et al.\u003c/em\u003e miR-142-3p Modulates Cell Invasion and Migration via PKM2-Mediated Aerobic Glycolysis in Colorectal Cancer. \u003cem\u003eAnalytical cellular pathology\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, 9927720, doi:10.1155/2021/9927720 (2021).\u003c/li\u003e\n\u003cli\u003eLuo, Y.\u003cem\u003e et al.\u003c/em\u003e miR-137 represses migration and cell motility by targeting COX-2 in non-small cell lung cancer. \u003cem\u003eTranslational cancer research\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 3803-3813, doi:10.21037/tcr-22-2177 (2022).\u003c/li\u003e\n\u003cli\u003eBi, W. P., Xia, M. \u0026amp; Wang, X. J. miR-137 suppresses proliferation, migration and invasion of colon cancer cell lines by targeting TCF4. \u003cem\u003eOncology letters\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 8744-8748, doi:10.3892/ol.2018.8364 (2018).\u003c/li\u003e\n\u003cli\u003eWang, D., Zhang, X., Lu, Y., Wang, X. \u0026amp; Zhu, L. Hypoxia inducible factor 1alpha in hepatocellular carcinoma with cirrhosis: Association with prognosis. \u003cem\u003ePathology, research and practice\u003c/em\u003e \u003cstrong\u003e214\u003c/strong\u003e, 1987-1992, doi:10.1016/j.prp.2018.09.007 (2018).\u003c/li\u003e\n\u003cli\u003eFeng, W.\u003cem\u003e et al.\u003c/em\u003e HIF-1alpha promotes the migration and invasion of hepatocellular carcinoma cells via the IL-8-NF-kappaB axis. \u003cem\u003eCellular \u0026amp; molecular biology letters\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 26, doi:10.1186/s11658-018-0077-1 (2018).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cdiv\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"335\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eCorrelation between FOXO expression and clinicopathological features in 37 patients with the recurrence of HCC after hepatectomy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"21\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"NaN%\" height=\"21\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.47761194029851%\" rowspan=\"2\"\u003e\n \u003cp\u003eCharacteristics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.38805970149254%\" rowspan=\"2\"\u003e\n \u003cp\u003eFOXO positive (n/N) (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.134328358208956%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"21\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"NaN%\" height=\"21\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.47761194029851%\"\u003e\n \u003cp\u003eTNM stage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.38805970149254%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.134328358208956%\"\u003e\n \u003cp\u003e0.189\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"16\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.47761194029851%\"\u003e\n \u003cp\u003eI-II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.38805970149254%\"\u003e\n \u003cp\u003e7/13 (54)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.134328358208956%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"0%\" height=\"16\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.47761194029851%\"\u003e\n \u003cp\u003eIII-IVa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.38805970149254%\"\u003e\n \u003cp\u003e18/24 (75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.134328358208956%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"0%\" height=\"16\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.47761194029851%\"\u003e\n \u003cp\u003eTumor diameter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.38805970149254%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.134328358208956%\"\u003e\n \u003cp\u003e0.923\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"16\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.47761194029851%\"\u003e\n \u003cp\u003e\u0026lt; 5 cm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.38805970149254%\"\u003e\n \u003cp\u003e15/22 (68)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.134328358208956%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"0%\" height=\"16\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.47761194029851%\"\u003e\n \u003cp\u003e\u0026ge; 5 cm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.38805970149254%\"\u003e\n \u003cp\u003e10/15 (67)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.134328358208956%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"0%\" height=\"16\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.47761194029851%\"\u003e\n \u003cp\u003eTumor number\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.38805970149254%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.134328358208956%\"\u003e\n \u003cp\u003e0.523\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"16\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.47761194029851%\"\u003e\n \u003cp\u003e\u0026lt; 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.38805970149254%\"\u003e\n \u003cp\u003e21/32 (66)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.134328358208956%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"0%\" height=\"16\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.47761194029851%\"\u003e\n \u003cp\u003e\u0026ge; 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.38805970149254%\"\u003e\n \u003cp\u003e4/5 (80)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.134328358208956%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"0%\" height=\"16\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.47761194029851%\"\u003e\n \u003cp\u003eVascular invasion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.38805970149254%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.134328358208956%\"\u003e\n \u003cp\u003e0.007*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"16\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.47761194029851%\"\u003e\n \u003cp\u003ePresent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.38805970149254%\"\u003e\n \u003cp\u003e18/21 (86)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.134328358208956%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"0%\" height=\"16\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.47761194029851%\"\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.38805970149254%\"\u003e\n \u003cp\u003e7/16 (44)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.134328358208956%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"0%\" height=\"16\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.47761194029851%\"\u003e\n \u003cp\u003eSerum AFP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.38805970149254%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.134328358208956%\"\u003e\n \u003cp\u003e0.114\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"16\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.47761194029851%\"\u003e\n \u003cp\u003e\u0026le; 20 \u0026mu;g/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.38805970149254%\"\u003e\n \u003cp\u003e6/12 (50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.134328358208956%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"0%\" height=\"16\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.47761194029851%\"\u003e\n \u003cp\u003e\u0026gt; 20 \u0026mu;g/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.38805970149254%\"\u003e\n \u003cp\u003e19/25 (76)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.134328358208956%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"0%\" height=\"16\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.47761194029851%\"\u003e\n \u003cp\u003eHistological differentiation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.38805970149254%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.134328358208956%\"\u003e\n \u003cp\u003e0.04*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"16\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.47761194029851%\"\u003e\n \u003cp\u003eGood\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.38805970149254%\"\u003e\n \u003cp\u003e3/8 (38)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.134328358208956%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"0%\" height=\"16\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.47761194029851%\"\u003e\n \u003cp\u003eModerate and poor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.38805970149254%\"\u003e\n \u003cp\u003e22/29 (76)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.134328358208956%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"16\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\"\u003e\n \u003cp\u003e\u0026chi;2 test; TNM: Tumor, node and metastasis; AFP: \u0026alpha; fetoprotein; *, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"16\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\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":"Hepatocellular carcinoma, Rac1, FOXO6, target gene, transcriptional regulation","lastPublishedDoi":"10.21203/rs.3.rs-3782217/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3782217/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRac1 activation is a common occurrence in various tumors and is often associated with poor prognosis, underscoring the potential therapeutic value of targeting the Rac1 pathway. Therefore, selectively inhibiting the heightened Rac1 activity in tumor cells may represent an innovative approach to cancer treatment. In this study, we found the increase in Rac1 expression contributes to heightened Rac1 activity and enhanced migration of HCC cells. Notably, our investigations identified FOXO6, rather than HIF-1α, Smad7, miR-142-3p, or miR-137, as the mediator of Rac1 expression. FOXO6 exhibits transcriptional activation and correlates with the early recurrence of HCC following hepatectomy. The transcriptional activation of the Rac1 gene hinges on a FOXO-binding sequence in the Rac1 gene promoter. FOXO6 was found to directly bind to this sequence both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Ultimately, Rac1 operates downstream of the FOXO6-dependent pro-migration signaling cascade. Our findings illuminate the direct role of FOXO6 in mediating the upregulation of Rac1 expression and activity in HCC cells. This discovery unveils a differentially activated FOXO6/Rac1 pathway in liver cancer, thereby positioning FOXO6 as a potential therapeutic target for liver cancer treatment, offering the prospect of mitigating excessive side effects.\u003c/p\u003e","manuscriptTitle":"FOXO6 specifically mediates overactivation of Rac1 in hepatocellular carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-18 07:25:28","doi":"10.21203/rs.3.rs-3782217/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":"cedcde2b-7a5f-4212-8a03-2a362bcff92d","owner":[],"postedDate":"January 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-05T16:18:47+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-18 07:25:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3782217","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3782217","identity":"rs-3782217","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}