Mechanistic insights into hypoxia-induced TCF7L2 upregulation and its oncogenic effects on colorectal cancer

Mechanistic insights into hypoxia-induced TCF7L2 upregulation and its oncogenic effects on colorectal cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mechanistic insights into hypoxia-induced TCF7L2 upregulation and its oncogenic effects on colorectal cancer KANG TANG, YONG Cheng, JIANPING GONG, YANG LI This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7002021/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 Hypoxia plays a crucial role in the advancement of colorectal cancer (CRC); however, the downstream mechanisms facilitated by hypoxia-inducible factor 1α (HIF-1α) remain incompletely understood. This study employed in vitro and in vivo models to investigate the role of transcription factor 7-like 2 (TCF7L2) under hypoxic conditions in CRC. Utilizing reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, we observed an upregulation of TCF7L2 mRNA and protein expression in Caco-2 and HCT-116 CRC cell lines under hypoxia. Functional assays, including CCK-8, colony and sphere formation, Transwell, flow cytometry, and xenograft tumor models, provided evidence that the knockdown of TCF7L2 leads to the suppression of CRC cell proliferation, the induction of apoptosis, cell cycle arrest at the G0/G1 phase, and a decrease in migration and invasion capabilities. Furthermore, it inhibited epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) characteristics in vitro , while also reducing tumor growth in vivo . Mechanistically, chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (co-IP) assays have elucidated that the expression of TCF7L2 induced by hypoxia is dependent on HIF-1α, which directly binds to hypoxia response elements (HREs) within the TCF7L2 promoter. Additionally, Western blot and experiments employing the PI3K inhibitor LY294002 have demonstrated that TCF7L2 activates the PI3K/AKT signaling pathway, thereby facilitating the proliferation of CRC cells. A clinical analysis of 104 CRC specimens, utilizing immunohistochemistry (IHC) and RT-qPCR, revealed that elevated expression levels of TCF7L2 were significantly associated with advanced T stage, metastasis, and unfavorable prognosis. Spearman correlation analysis confirmed a positive relationship between the expressions of TCF7L2 and HIF-1α, while Kaplan-Meier survival analysis demonstrated that their co-expression was predictive of reduced overall survival. Collectively, these findings position TCF7L2 as a critical downstream effector of HIF-1α in CRC, underscoring its potential as a therapeutic target for addressing hypoxic tumor microenvironments. transcription factor 7-like 2 hypoxia colorectal cancer epithelial-mesenchymal transition cancer stem cell Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Colorectal cancer (CRC) is one of the most prevalent solid malignancies and the third leading cause of tumor-related mortality worldwide, with the third highest disease-specific death rate (1). Multiple treatments, such as gene therapy, total mesorectal excision, neoadjuvant chemotherapy or chemoradiotherapy, and multidisciplinary team management, have been developed and widely applied in the clinic in recent decades (2). However, combination therapy, including targeted therapy for advanced CRC, has limited effectiveness, as patients with advanced CRC still exhibit poor survival, with a 5-year survival rate of ~12% (3,4). In addition, the molecular mechanisms underlying CRC initiation, progression and metastasis are yet to be fully elucidated. Therefore, identifying the precise biological mechanisms of CRC and exploring novel effective molecular therapeutic targets is imperative to improve survival in patients with CRC. Hypoxia is one of the most common and critical characteristics of the microenvironment during solid tumor progression (5). It has been reported that hypoxia-inducible factors (HIFs) participate in proliferation, inflammation, angiogenesis, invasion and distant metastasis in various types of solid carcinoma (6). Cancer cells undergo a series of complex adaptive changes in response to the hypoxic cellular microenvironment, including cell energy metabolism, proliferation, apoptosis, invasion and angiogenesis. HIF-1, a heterodimer consisting of α and β subunits, is a primary regulator of cellular responses to hypoxia during tumor progression. Several studies have shown that HIF-1α evades degradation under a hypoxic microenvironment by inhibiting prolyl hydroxylase domain hydroxylation (7-8). HIF-1α combines with the HIF-1β subunit to form a heterodimer and then translocates from the cytoplasm into the nucleus, where it interacts with specific DNA sequences containing hypoxia response elements (HREs), and mediates the transcription and expression of multiple downstream target genes (9). To date, >100 target genes downstream of HIF have been discovered, most of which are involved in the metastasis cascade, including epithelial-mesenchymal transition (EMT), extracellular matrix, enhanced tumor cell motility and angiogenesis (10). Clinical studies have demonstrated that elevated expression of HIF-1α is positively associated with poor prognosis in most solid carcinoma types, including breast cancer (11), hepatocellular carcinoma (12), ovarian cancer (13), esophageal cancer (14) and CRC (15). Transcription factor 7-like 2 (TCF7L2) directly regulates genes involved in metabolism and cell cycle control within adipocytes (16). Notably, TCF7L2 has been reported to be expressed in various types of non-mineralizing soft cancerous tissues, including in colon, esophageal, lung, skin and stomach cancer, suggesting that TCF7L2 may have an essential role in the carcinogenesis of malignant tumors (17-19). It has also been reported that the TCF7L2 gene is associated with type 2 diabetes (20) and is inversely associated with prostate cancer (21). Meanwhile, the association between TCF7L2 and colon cancer has been demonstrated in nondiabetic participants (22). Moreover, a previous study revealed that TCF7L2 may be upregulated in mammary epithelial cell-derived organoids and involved in EMT. Notably, abnormal cellular expression of the TCF7L2 protein has been shown to be positively associated with increased expression levels of HIF-1α (23). Similarly, the hypoxic microenvironment can increase the expression of TCF7L2 in clear cell renal cell carcinoma (ccRCC) in a HIF-2α-dependent manner, and hypoxia-regulated TCF7L2 high expression participates in ccRCC tumor survival and distant metastasis (24). However, few studies have explored the function of TCF7L2 in CRC (25). This study seeks to evaluate the biological functions, clinical relevance, and underlying mechanisms of TCF7L2 in CRC. The research specifically aims to elucidate the regulatory role of TCF7L2 in key cellular processes, including proliferation, apoptosis, EMT, and cancer stemness maintenance, under hypoxia and normoxia. Additionally, the study examines the clinical significance of TCF7L2 expression in CRC by analyzing its association with tumor stage, metastasis, and patient prognosis. Mechanistically, the investigation focuses on the HIF-1α-dependent upregulation of TCF7L2, emphasizing HIF-1α-mediated transcriptional activation and its interaction with the TCF7L2 promoter, as well as the subsequent downstream signaling pathway. Materials and methods Clinical specimens and cell culture. A total of 104 clinical CRC samples and adjacent non-tumor samples were obtained from the Department of Gastrointestinal Surgery, The Second Affiliated Hospital of Chongqing Medical University (Chongqing, China) between January 2009 and December 2013. No patients received preoperative radiotherapy or chemotherapy before surgery. All patients provided written informed consent. The inclusion criteria were as follows: i) A diagnosis of colorectal adenocarcinoma confirmed through pathological examination, with patients having undergone radical resection for CRC; ii) absence of preoperative chemoradiotherapy. The exclusion criteria were as follows: i) Preoperative pathological diagnosis of mucinous adenocarcinoma, signet ring cell carcinoma, adenosquamous carcinoma, medullary carcinoma, undifferentiated carcinoma or carcinosarcoma; ii) incomplete case records; iii) patients who underwent palliative surgery for CRC. The present study was approved by the Ethical Committee of The Second Affiliated Hospital of Chongqing Medical University (ethical approval no. 2023 − 326). CRC cell lines Caco-2 (cat. no. HTB-37), HCT-116 (cat. no. CCL-247), HT-29 (cat. no. HTB-38), LoVo (cat. no. CCL-229), SW480 (cat. no. CCL-228) and SW620 (cat. no. CCL-227), and the human immortalized normal small intestine epithelium cell line HIEC-6 (cat. no. CRL-3266) were obtained from the American Type Culture Collection. All of the cell lines used in the present study were obtained from the Central Laboratory of Chongqing Medical University, which were purchased from ATCC. The cells were genotyped by short tandem repeat analysis and were tested for Mycoplasma before use. All CRC cell lines were cultured in Leibovitz’s L-15 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) in a 21% O 2 , 5% CO 2 incubator at 37˚C. HIEC-6 cells were cultured in Opti-MEM I Reduced Serum Medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 4% FBS and 10 ng/ml epidermal growth factor (EGF; MilliporeSigma) in a 21% O 2 , 5% CO 2 incubator at 37˚C. To mimic the hypoxic microenvironment, cells were cultured in a hypoxic cell incubator (Thermo Fisher Scientific, Inc.) supplemented with 1% O 2 , 5% CO 2 and 94% N 2 . RNA interference. Transduction-ready TCF7L2 short hairpin RNA (shRNA) (h) Lentiviral Particles (cat. no. sc-43525-V) and HIF-1α shRNA (h) Lentiviral Particles (cat. no. sc-35561-V) (both from Santa Cruz Biotechnology, Inc.) were transduced into both Caco-2 and HCT-116 cells to establish stable knockdown of TCF7L2 or HIF-1α. Briefly, CRC cell lines (5x10 4 /well) were plated in a 6-well plate 24 h before lentiviral particle infection. The thawed lentiviral particles were then transduced into cells overnight with a multiplicity of infection of 4, alongside 5 µg/ml polybrene (Santa Cruz Biotechnology, Inc.). Stable clones expressing TCF7L2 or HIF-1α shRNA were selected and maintained using 10 µg/ml puromycin dihydrochloride (MilliporeSigma) for 14 days and harvest immediately for subsequent experiments. Control shRNA Lentiviral Particles (cat. no. sc-108080; Santa Cruz Biotechnology, Inc.) were transduced into cells as a negative control. In addition, TCF7L2 Lentiviral Activation Particles (h) (cat. no. sc-400607-LAC; Santa Cruz Biotechnology, Inc.) were transduced into Caco-2 and HCT-116 cells to establish stable overexpression of TCF7L2. Control Lentiviral Activation Particles (cat. no. sc-437282; Santa Cruz Biotechnology, Inc.) were transduced into cells as a negative control. The specific operation procedure and method were applied as previously described ( 26 ). RNA extraction and reverse transcription-quantitative PCR (RT-qPCR). Total RNA was isolated from tissues or cultured CRC cells using RNAiso plus (Takara Bio, Inc.) according to the manufacturer’s protocol. cDNA was then synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to manufacturer’s protocol. qPCR analyses were conducted by using SsoFast EvaGreen Supermix (Bio-Rad) in Bio-Rad CFX96 system as previously described ( 26 ). Thermocycling conditions included an initial enzyme activation at 95˚C for 30 sec, followed by 40 cycles of denaturation at 95˚C for 5 sec, annealing/extension at 60˚C for 5 sec. The relative expression levels of mRNA were calculated according to the 2 −ΔΔCq method, where: ΔΔCq = Δ Cq (sample) - ΔCq (control) ( 27 ). The primer sequences used for qPCR analysis are listed in Table SI. All reactions were performed in triplicate. Western blot analysis. Proteins were extracted from frozen tissues or cultured CRC cells using RIPA lysis buffer containing phosphatase inhibitor (MilliporeSigma). Subsequently, Protein concentrations were quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific) according to manufacturer’s protocol prior to loading ~ 20 µg protein samples were separated electrophoresis on 10% by SDS-PAGE gels, transferred onto polyvinylidene fluoride membranes and blocked with 3% bovine serum albumin (MilliporeSigma) diluted with Tris-buffered saline containing 0.1% Tween 20 solution at room temperature for 1 h. The membranes were then incubated at 4˚C overnight with the primary antibodies. The information on all primary antibodies used in the present study is provided in Table SII. Subsequently, membranes were incubated at room temperature for 1 h with a horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) secondary antibody (1:2,000 dilution; cat. no. sc-2301; Santa Cruz Biotechnology) or HRP-conjugated anti-mouse IgG HRP secondary antibody (1:2,000 dilution; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.). Protein bands were visualized using the ECL Prime Western Blotting Detection Reagent (Amersham). Densitometric analysis of immunoblot bands was performed using Quantity One (Bio-Rad Quantity One version 4.6.2). The specific experimental methods were conducted as previously described ( 26 ). Cell proliferation, colony formation and sphere formation assays. The cell proliferation assay was carried out using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Inc.) at indicated time points (6, 24, 48, 72 and 96 hours) according to the manufacturer’s protocol. Colony formation assays were performed as follows. Briefly, 1x10 3 cells/well were seeded into a 6-well plate and were cultured in complete cell medium for 12 days. Colonies in each group were then fixed and stained with a Diff-Quick kit (Sysmex Corporation), cells were fixed with the methanol-based solution (Component A ), at room temperature (RT) for 1 minute. Following fixation, colonies were stained sequentially with the Diff-Quick Stain Solution I (Component B, eosinophilic dye) at RT for 1 minute and Diff-Quick Stain Solution II (Component C, basophilic dye) at RT for 1 minute. A colony was defined as consisting of ≥ 50 cells. Plating efficiency (PE) was calculated using the following formula: PE (%) = number of colonies formed/number of cells seeded x 100. For the sphere formation assay, 1x10 4 cells were cultured on low-adherence plates in 10 ml serum-free medium supplemented with EGF (10 ng/ml, Invitrogen), basic fibroblast growth factor (20 ng/ml, Invitrogen), 4% B27 (Invitrogen) and 4 µg/ml insulin (MilliporeSigma). A light microscope (Olympus Microscope) was used to visualize and quantify tumor spheres under bright-field conditions. Sphere formation efficiency (SFE) was computed using the following formula: SFE (%) = number of spheres formed at day 14/number of cells seeded x 100. The specific experimental methods were carried out as previously described ( 26 ). Transwell migration and invasion assays. In vitro migration (using Transwell plates that were not pre-coated with Matrigel) and invasion (using Transwell plates that were pre-coated with Matrigel at 37°C for 2 hours) assays were performed using 24-well Transwell plates (diameter, 6.5 mm; pore size, 8 µm; Corning, Inc.). Briefly, CRC cells (2x10 5 ) were plated into the upper chamber was filled with serum-free medium, while the lower chamber contained complete medium supplemented with 10% fetal bovine serum (FBS) to drive cell migration and invasion. Transwell plates were incubated at 37˚C for 24 h (migration) or 30 h (invasion). Finally, migratory or invasive cells that were attached to the lower chamber were gently washed with PBS, fixed and stained with a Diff-Quick stain kit (Sysmex Corporation), the membrane was fixed with Component A for 1 minute at RT. Following fixation, the membrane was sequentially stained using the Solution I (eosin-based stain) for 1 minute at RT, followed by Solution II (methylene blue-based stain) for 1 minute at RT. Cellular staining was visualized using a bright field compound light microscope (Olympus). The specific experimental methods were conducted as previously described ( 26 ). Cell apoptosis and cell cycle analyses. Cell apoptosis analysis was performed using the Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit (BD Biosciences) according to the manufacturer’s protocol. Briefly, CRC cells (5x10 5 cells) in each indicated group were harvested and washed three times in ice-cold PBS. Cells were then transferred into 500 µl Annexin V-binding buffer with 5 µl Annexin V-FITC and 5 µl (50 µg/ml) propidium iodide (PI), and were incubated at room temperature for 15 min in the dark. Flow cytometric analysis (BD FACSCalibur, BD Biosciences) was applied to quantify the percentage of apoptosis in CRC cells in each indicated group, and the data were analyzed with FlowJo software (version 10.7.2, BD Biosciences). For cell cycle analysis, 5x10 5 cells in each indicated group were harvested after treatment, washed with PBS, fixed in 70% ice-clod ethanol at 4˚C overnight, treated with RNaseA (50 µg/ml) and 0.1% Triton X-100, and stained with PI (40 µg/ml) at room temperature for 30 min in the dark, followed by fluorescence-activated cell sorting (FACS) analysis (BD FACSCalibur, BD Biosciences). To assess cell cycle distribution, DNA content was determined using ModFit LT 3.0 software (Verity Software House, Inc.). The specific experimental methods were performed as previously described ( 26 ). Xenograft mouse assay. A total of 20 4-6-week-old male BALB/c-nu/nu mice (weighing 18–20 g each) were purchased from the Laboratory Animal Center of Chongqing Medical University and were used for xenograft tumorigenicity assays. Mice were randomly allocated to each indicated group (n = 5/group) and were housed in specific pathogen-free facilities under standard housing conditions with a 12-h light/dark cycle. Mice were provided ad libitum access to food and water, with housing conditions maintained at 22 ± 2°C and 50% ± 5% humidity. The animal experiment was designed to be a single-blind trial. Briefly, a final concentration of 2x10 6 Caco-2 or HCT-116 cells in each group was suspended in 200 µl medium with Matrigel (1:1) and subcutaneously injected into the right rear flank of nude mice. Mice were randomly divided into four groups as follows: Caco-2 Nc-shRNA (Caco-2 cells transfected with negative control shRNA), Caco-2 TFC7L2-shRNA (Caco-2 cells transfected with TCF7L2 shRNA), HCT-116 Nc-shRNA (HCT-116 cells transfected with negative control shRNA), HCT-116 TFC7L2-shRNA (HCT-116 cells transfected with TCF7L2 shRNA). Tumor size was measured twice weekly using calipers. Mice were sacrificed 6 weeks after injection by cervical dislocation under ether anesthesia. Humane endpoints were predefined as tumor volume exceeding 2000 mm³. The tumor volume was calculated using the following formula: Tumor volume (mm 3 ) = 0.5 x [length (mm)] x [width (mm) 2 ]. All animal experiments were approved by the Animal Ethics Committee of Chongqing Medical University (ethical approval no. 2023 − 326). Glucose assay. The glucose concentration in the culture supernatant was determined using the Glucose Assay Kit-WST (Dojindo Laboratories, Inc.). Briefly, prior to the experiment, standard curves were drawn with a standard glucose solution (0-0.5 µM). Cells (5x10 5 cells/well) were seeded in a 6-well microplate and were cultured at 37˚C for 24 h. Subsequently, 100 µl supernatant was transferred to a 1.5-ml microtube and diluted 40-fold with double distilled water to generate the sample solution. Approximately 50 µl sample solution and 50 µl working solution were then added to a 96-well microplate, which was incubated at 37˚C for 30 min. The absorbance of each well was measured at 450 nm using a microplate reader and glucose concentration in each sample solution was calculated using the aforementioned standard curve. Flow cytometry and magnetic cell sorting. Briefly, ~5x10 5 CRC cells in each indicated group were suspended in 200 µl cell staining buffer (cat. no. 420201; BioLegend, Inc.) and labeled with 10 µl FITC-conjugated anti-human CD44 (cat. no. 338804; BioLegend, Inc.) or phycoerythrin (PE)-conjugated anti-human CD133 (cat. no. 372804; BioLegend, Inc.) at 4˚C for 15 min in the dark. Samples were then centrifuged at 350 x g for 5 min at 4˚C and washed twice with 2 ml cell staining buffer. Subsequently, CD44 or CD133 expression was examined by flow cytometry (BD FACSCalibur, BD Biosciences), the data were analyzed with FlowJo software (version 10.7.2, BD Biosciences). FACS of CD44 + /CD133 + double-positive cells and CD44 − /CD133 − double-negative HCT-116 cells was performed using a FACS ARIA II high-speed cell sorter (BD Biosciences) according to the manufacturer’s instructions. Chemical and chemotherapy drug treatment. The PI3K inhibitor LY294002 (MilliporeSigma) was added to the cell culture medium at 37°C and a final concentration of 10 µM 24 h prior to the start of experiments assessing TCF7L2-mediated cell proliferation and PI3K/AKT signaling activation. The working concentration and processing time of LY294002 were selected based on the manufacturer’s protocol. The chemoresistance of CRC cells to 5-fluorouracil (5-FU) or oxaliplatin (LOHP) was analyzed using the CCK-8. The concentration gradient of 5-FU and LOHP was set according to a previous publication ( 26 ). Briefly, 5x10 3 cells (per well) in each indicated group (Normoxia, hypoxia, hypoxia Negative Control (NC)-sh, hypoxia TCF7L2-sh) were seeded into a 96-well plate and incubated with different concentrations (0, 1, 2, 4, 6, 8,10, 50 and 100 µg/ml) of 5-FU or LOHP for 72 h at 37˚C. Subsequently, the medium was replaced with fresh medium containing 100 µl CCK-8 solution. Optical density values were determined at 450 nm using a microplate reader (Sanyo). Drug response curves and half-maximal inhibitory concentration (IC₅₀) values were generated using GraphPad Prism 8 software (GraphPad Software) via nonlinear regression analysis of dose–response data. Co-immunoprecipitation assay. Co-immunoprecipitation was carried out using the Dynabeads Protein G Immunoprecipitation Kit (cat. no. 10004D, Thermo Fisher Scientific, Inc.), according to the manufacturer’s instructions. Antibody-conjugated magnetic beads were generated by coupling 5 µg anti-HIF-1α (1:150 dilution; cat. no. Ab1; Abcam) or anti-TCF7L2 (1:30 dilution; cat. no. sc-166699; Santa Cruz Biotechnology, Inc.) antibodies with 50 µl (1.5 mg) magnetic beads, followed by resuspension in 200 µl antibody binding and elution buffer, and incubation with gentle rotation at room temperature for 15 min. Following incubation, 500 µl cell lysates containing the antigen were gently pipetted to resuspend the pre-prepared magnetic bead-antibody complex. Cell lysates were further incubated with rotation at 4˚C overnight to allow the antigen to bind to the magnetic bead-antibody complex. Finally, the magnetic bead-antibody-antigen complex was eluted in 20 µl elution buffer, and 20 µl eluted samples were mixed with 20 µl protein loading buffer. The mixture was heated at 95˚C for 5 min for SDS-PAGE and western blot analysis as aforementioned. Normal mouse IgG (1:30 dilution; cat. no. CST-7076S; Cell Signaling Technology, Inc.) was used as a negative control antibody. The specific experimental methods were conducted as previously described ( 26 ). Chromatin immunoprecipitation (ChIP) assay. The promoter region of TCF7L2 was identified using publicly available online resources (NC_000010.11:c112950191-112950182, https://www.ncbi.nlm.nih.gov/nuccore/NC_000010.11?from=112950182&to=112950191&strand=true ). The analysis concentrated on the first 1,000 base pairs upstream of the transcription start site. Utilizing computational screening with algorithms from the JASPAR database ( http://jaspar.genereg.net/ ), HREs or HIF-1 binding sites within the promoter region were identified, specifically within the 1,000 base pairs upstream of the TCF7L2 transcription start site (TSS). The ChIP assay was performed using the Simple ChIP Plus Enzymatic Chromatin IP Kit (Agarose Beads; cat. no. CST-9005; Cell Signaling Technology, Inc.) according to manufacturer’s protocol. Briefly, ~ 5 µg anti-HIF-1α (1:150 dilution; cat. no. Ab1; Abcam) or negative control mouse IgG antibodies (1:30 dilution; cat. no. CST-7076S; Cell Signaling Technology, Inc.) were used for immunoprecipitation in ChIP reactions. Quantitative real-time PCR (qPCR) was performed immediately following the ChIP assay. The primers designed for ChIP assays are listed in Table SI. The specific experimental methods were performed as previously described ( 26 ). Immunohistochemistry (IHC). IHC was conducted on paraformaldehyde-fixed, paraffin-embedded CRC patients tumor tissue sections. Tumor tissues were fixed in 4% paraformaldehyde at room temperature for 24 hours, embedded in paraffin, and serially sectioned into 4-µm-thick slices. Sections were dewaxed with xylene and rehydrated through a gradient ethanol series (100%, 95%, 85%, 70%, v/v). Endogenous peroxidase activity was quenched by incubation with 3% hydrogen peroxide (H₂O₂) for 10 minutes at room temperature. Antigen retrieval was performed via microwave heating in 0.01 mol/L sodium citrate buffer (pH 6.0) at 95°C for 15 minutes, followed by cooling to room temperature. Sections were blocked with 5% bovine serum albumin (BSA; MilliporeSigma) in phosphate-buffered saline (PBS) for 20 minutes at room temperature to reduce nonspecific binding. Primary antibodies against HIF-1α (1:100 dilution; cat. no. ab1, Abcam) or TCF7L2 (1:50 dilution; cat. no. sc166699, Santa Cruz Biotechnology, Inc.) were applied and incubated overnight at 4°C in a humidified chamber. After washing with PBS, sections were probed with horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody (1:2,000 dilution; cat. no. sc-2005, Santa Cruz Biotechnology, Inc.) for 20 minutes at room temperature. Color development was achieved using 3,3'-diaminobenzidine (DAB; cat. no. 3468, Dako) according to the manufacturer’s instructions. Slides were counterstained with hematoxylin, dehydrated, cleared, and coverslipped. Staining was visualized and imaged under a bright-field light microscope (Olympus). Quantification of positive cell percentages was performed using ImageJ 1.48 software (National Institutes of Health), with mean values calculated from three independent fields per section. Protein expression levels were assessed independently by two investigators in collaboration with a pathologist, all of whom were blinded to the clinical information of the patients. The methodology for evaluating IHC staining results was structured as follows: The intensity score was categorized as 0 for no staining, 1 for weak staining, 2 for moderate staining and 3 for strong staining. The percentage score was defined as Score 0 for staining covering 0–4% positive cells, Score 1 for 5–24% positive cells, Score 2 for 25–49% positive cells, Score 3 for 50–74% positive cells, and Score 4 for 75–100% positive cells. The final IHC score was calculated by multiplying the intensity score by the percentage score. High expression levels for HIF-1α and TCF4/TCF7L2 were identified as having final IHC scores of 1 + and 3+, respectively. Statistical analysis. Data are presented as the mean ± SD from three independent experimental repeats. All statistical analyses were performed using GraphPad Prism 8.0 (Dotmatics) and SPSS version 22.0 (IBM Corp.). The unpaired Student’s t-test was used for comparisons between two independent groups, whereas a paired two-tailed Student’s t-test was used to compare expression between CRC samples and adjacent non-tumor tissues. One-way analysis of variance was applied for multiple group comparisons followed by Tukey's multiple comparison test. Correlation analyses between groups were assessed using Spearman rank correlation. Categorical variables were assessed using the χ 2 test or Fisher’s exact test. The Kaplan-Meier log-rank test was used to analyze the overall survival curve data. P < 0.05 was considered to indicate a statistically significant difference. Results TCF7L2 expression is upregulated in CRC cell lines under hypoxia. TCF7L2 mRNA and protein expression levels were first evaluated in Caco-2, HCT-116, HT-29, LoVo, SW480 and SW620 cells. HIEC-6 cells were selected as a normal small intestine epithelial cell line. The results showed that the mRNA levels of TCF7L2 were markedly upregulated in CRC cell lines compared with those in the HIEC-6 cell line (Fig. 1 A). Western blot analysis revealed a similar trend in TCF7L2 protein expression levels in CRC cell lines (Fig. 1 B). Hypoxia is one of the most critical niches during solid tumor progression ( 28 ). Subsequently, the mRNA and protein levels of TCF7L2 were revealed to be significantly upregulated in Caco-2 and HCT-116 cells under hypoxic conditions compared with under normoxia (Fig. 1 C and D). Given that TCF7L2 was significantly upregulated in these two cell lines under hypoxic conditions, these two cell lines were selected for subsequent experiments to evaluate the biological impacts and investigate the mechanisms of TCF7L2 knockdown in colorectal cancer cells under hypoxia. TCF7L2 promotes CRC cell proliferation, migration, invasion and EMT in the presence of hypoxia. Subsequently, stable TCF7L2 knockdown was induced in Caco-2 and HCT-116 cell lines using shRNA lentiviral particles to further elucidate the biological roles of TCF7L2 in CRC cell lines. RT-qPCR and western blot analysis were performed post-transduction with TCF7L2-shRNA to examine the mRNA and protein expression levels of TCF7L2, respectively. The results showed that cells with TCF7L2 knockdown had significantly lower levels of TCF7L2 mRNA and protein than negative control cells (Fig. 2 A and B). The role of TCF7L2 in Caco-2 and HCT-116 cell proliferation was determined in vitro using a CCK-8 assay. The results revealed that the proliferation rates of Caco-2 and HCT-116 cells were significantly increased under hypoxic conditions; however, TCF7L2 knockdown markedly inhibited hypoxia-induced hyper-proliferation (Fig. 2 C and D). Furthermore, it was observed that the proliferative capacity of HCT-116 and Caco-2 cells was markedly reduced following the knockdown of TCF7L2 under normoxic conditions (Fig. S1A and B). Given that TCF7L2 serves an important role in CRC cell proliferation in vitro , the impact of TCF7L2 on the tumorigenicity of CRC cells in vivo was investigated. The results revealed that TCF7L2 knockdown significantly suppressed tumor growth in vivo (Fig. 2 G). Taken together, these results indicates that TCF7L2 may have a vital role in cancer progression both in vitro and in vivo . Glucose is essential for cell proliferation, growth and survival. Thus, the Glucose Assay Kit-WST, which enables the quantitation of glucose as a substrate in energy metabolism, was utilized to assess the effects of TCF7L2 on cancer metabolism. It was revealed that the levels of glucose were decreased in the Caco-2 and HCT-116 cell supernatant after hypoxic stimulation; however, TCF7L2 knockdown reversed this trend (Fig. 2 H), thus suggesting that TCF7L2 may affect the efficiency of glucose utilization in CRC cells. Metastasis is considered a crucial event during CRC development. Subsequently, Transwell migration and invasion assays were employed to investigate the role of TCF7L2 in hypoxia-induced CRC metastasis capability. The results showed that Caco-2 and HCT-116 cells exhibited significantly enhanced cell migration and invasion under hypoxic conditions; however, TCF7L2 knockdown blocked Caco-2 and HCT-116 cell migration and invasion (Fig. 2 I and J). MMPs have been shown to serve an important role in tumor invasion and metastasis( 29 ), and MMP2 and MMP9 are critical MMPs that regulate cell migration and invasion. In the present study, MMP9, but not MMP2, was highly expressed in Caco-2 and HCT-116 cells under hypoxic conditions; however, TCF7L2 knockdown decreased the expression levels of MMP9 (Fig. 2 K). Accumulating evidence has confirmed that hypoxia has an important role in cancer metastasis and EMT. To further explore the molecular mechanism underlying the role of TCF7L2 in CRC development, the expression level of EMT markers and related transcription factors were detected. The results showed that hypoxia significantly downregulated the epithelial marker E-cadherin, but upregulated the mRNA expression levels of the mesenchymal markers N-cadherin and vimentin, as well as transcription factors Snail and Slug (Fig. 2 L and M). Conversely, TCF7L2 knockdown showed the opposite expression pattern of EMT-related genes in Caco-2 and HCT116 cells under hypoxic conditions. Consistent with the mRNA expression profiles, western blot analysis confirmed that hypoxia significantly promoted EMT progression in CRC cells; however, TCF7L2 knockdown partially reversed the role of hypoxia in EMT activation in Caco-2 and HCT116 cells (Fig. 2 N). Collectively, these results indicated that TCF7L2 may stimulate the survival and metastatic ability of CRC cell lines. TCF7L2 is involved in hypoxia-induced apoptosis resistance and cell cycle arrest in CRC cell lines. Apoptosis resistance serves an essential role during the response of tumor progression to hypoxia ( 30 ). Therefore, the apoptosis rate was detected to further investigate the role of TCF7L2 in Caco-2 and HCT-116 cells under hypoxic conditions. The results showed that the apoptosis rate was significantly decreased in Caco-2 and HCT-116 cells under hypoxic conditions; however, shRNA-mediated TCF7L2 knockdown significantly reduced hypoxia-induced apoptosis resistance (Fig. 3 A). Furthermore, cell cycle progression in Caco-2 and HCT-116 cells was examined by analyzing the distribution of cell cycle phases to further assess the role of TCF7L2 in the survival of CRC cells. The results revealed that the proportion of Caco-2 and HCT-116 cells in the G 0 /G 1 phase was significantly decreased under hypoxic conditions; however, TCF7L2 knockdown increased the proportion of cells in the G 0 /G 1 phase (Fig. 3 B). In addition, the protein expression levels of cyclin D1 and proliferating cell nuclear antigen (PCNA) were significantly increased in Caco-2 and HCT-116 cells under hypoxic conditions; conversely, TCF7L2 knockdown downregulated the expression levels of cyclin D1 and PCNA in these cells (Fig. 3 C). Taken together, these data suggested that TCF7L2 may promote CRC cell survival by inducing apoptosis resistance and cell cycle G 1 /S transition. TCF7L2 promotes CRC cell proliferation via the PI3K/Akt signaling pathway. Based on the aforementioned findings that the PI3K/Akt signaling pathway regulates the proliferation of CRC cells ( 31 ), the current study further explored the protein expression levels of p-PI3K p85 and p-AKT1 (Ser 473) in Caco-2 and HCT116 cells exposed to hypoxia. The results demonstrated that these proteins were elevated under hypoxia, whereas TCF7L2 knockdown reduced the hypoxia-induced changes to these protein levels (Fig. 3 D). To further confirm whether the PI3K/Akt signaling pathway participates in TCF7L2-induced cell proliferation, Caco-2 and HCT116 cells with successfully stable TCF7L2 overexpression were pretreated with the PI3K inhibitor LY294002 (10 µM) for 24 h to block the PI3K/Akt signaling pathway. It was observed that LY294002 treatment almost completely blocked the expression levels of p-PI3K p85 and p-AKT1 (Ser 473) (Fig. 3 E). Moreover, the enhanced cell proliferation detected following TCF7L2 overexpression was partially reversed by the inhibition of PI3K in CRC cells (Fig. 3 F). Collectively, these findings indicated that the PI3K/Akt signaling pathway may have a crucial role in TCF7L2-mediated proliferation of CRC cells. TCF7L2 is involved in hypoxia-induced chemoresistance in CRC cell lines. Studies have demonstrated that hypoxia influences the occurrence of chemoresistance in various types of solid tumors due to the activation of HIF-1α signaling ( 32 , 33 ). To investigate whether TCF7L2 contributed to the development of chemoresistance of CRC cells exposed to hypoxia in vitro , Caco-2 and HCT116 cells were treated with difference concentrations of 5-FU or LOHP, followed by calculation of the cell survival rate and IC 50 . To explore drug-induced apoptosis, Caco-2 and HCT116 cells were pretreated with 5 µg/ml 5-FU or LOHP for 72 h. The results showed that 5-FU or LOHP induced a dose-dependent decrease in the survival rate of Caco-2 and HCT116 cells. As expected, hypoxia stimulation triggered enhanced resistance of Caco-2 and HCT116 cells to 5-FU and LOHP intervention, with the IC 50 values of CRC cells exposed to hypoxia found to be significantly higher compared with those exposed to normoxia. Conversely, TCF7L2 knockdown in Caco-2 and HCT116 cells abrogated the hypoxia-induced chemoresistance (Fig. 4 A-D). Further analysis revealed that hypoxia caused a significant reduction in the drug-induced apoptosis of CRC cells, whereas TCF7L2 knockdown abolished this effect (Fig. 4 E). These findings indicated that TCF7L2 was involved in the regulation of chemosensitivity in CRC cells. TCF7L2 contributes to the maintenance of cancer stem cell (CSC)-like phenotypes of CRC cells. Given the potential role of TCF7L2 in enhancing chemoresistance in CRC cells, the current study subsequently explored whether TCF7L2 may regulate chemoresistance of CRC cells. Specifically, colony formation and sphere formation assays were conducted on Caco-2 and HCT116 cells exposed to hypoxia. The results indicated that hypoxia promoted the clonogenic ability of Caco-2 and HCT116 cells, whereas TCF7L2 knockdown induced the opposite effect (Fig. 5 A). Regarding tumor sphere formation, Caco-2 cells were unable to form spheres in low-adherent, serum-free and growth factor medium compared with the HCT116 cells (Fig. S1C). The results of the assay indicated that hypoxia caused significant sphere formation of HCT-116 cells, with similar findings observed in the secondary passage; conversely, TCF7L2 knockdown inhibited sphere formation and the propagation of HCT-116 cells cultured in hypoxic conditions (Fig. 5 B). Taken together, these results indicated that TCF7L2 participated in the regulatory effects of hypoxia on the CSC properties of CRC cells. To verify these observations, the expression of some common stemness-related markers were investigated by FACS (Fig. 5 C), RT-qPCR (Fig. 5 D) and western blotting (Fig. 5 E) in HCT116 cells. The results indicated that the expression of stemness-related markers was significantly elevated under hypoxia; however, TCF7L2 knockdown in HCT-116 cells decreased the expression levels of cancer stemness markers, including CD44, CD133, epithelial cell adhesion molecule (EpCAM), and Nanog. Notably, no significant difference was observed for aldehyde dehydrogenase 1 family member A1 (ALDH1A1) or OCT3/4. Taken together, these findings demonstrated that TCF7L2 was involved in the regulatory effects of hypoxia on the stemness properties of CRC cells. Next, the present study aimed to clarify whether TCF7L2 could independently affect the stemness of CRC cells. It has been reported that cellular surface proteins CD44 and CD133 are potential markers for specific subpopulations of HCT-116 cells ( 34 ). TCF7L2 knockdown was first established in bulk HCT-116 cells via lentiviral shRNA transduction. After selecting for stable knockdown, cells were labeled with CD44 and CD133 antibodies, and the CD44 + /CD133 + HCT-116 subpopulation was purified by FACS. The results of the present study showed that CD44 + /CD133 + HCT-116 subpopulations displayed enhanced SFE, whereas HCT-116 CD44 − /CD133 − subpopulations formed fewer spheres; as expected, TCF7L2 knockdown in HCT-116 CD44 + /CD133 + subpopulations induced a significant inhibition in SFE (Fig. 5 F). Next, flow cytometry (Fig. 5 G and H), RT-qPCR (Fig. 5 I) and western blotting (Fig. 5 J) were performed to further determine the effect of TCF7L2 on the expression of CSC markers in regulating cancer stemness. The expression levels of CD44, CD133, ALDH1A1, SRY-box 2 (SOX2), EpCAM, Nanog and OCT4, which are widely accepted biomarkers of stemness for most solid tumors, were analyzed. The findings indicated that, apart from SOX2, other stemness markers such as CD44, CD133, ALDH1A1, EpCAM, Nanog and OCT3/4 were markedly upregulated in CD44 + /CD133 + HCT-116 cells. Conversely, these markers exhibited a significant downregulation following the knockdown of TCF7L2. Collectively, these findings suggested that TCF7L2 could independently regulate the stemness of CRC cells. Hypoxia induces the upregulation of TCF7L2 via direct transcriptional stimulation of HIF-1α. Hypoxia has been shown to promote the development of CRC. In our previous study, it was shown that the mRNA and protein expression levels of TCF7L2 were elevated under hypoxia ( 21 ). Several other investigations have confirmed that HIF-1α and HIF-2α are key regulators of responses to hypoxia in solid tumors ( 35 , 36 ). Therefore, the current aimed to clarify the molecular mechanism by which hypoxia promoted TCF7L2 expression in CRC cells through RT-qPCR and western blotting in Caco-2 and HCT116 cells exposed to hypoxia. It was observed that the expression levels of HIF-1α were significantly enhanced, whereas those of HIF-2α were not altered, under hypoxia (Fig. 6 A and B), following 24 hours of hypoxic culture (1% O₂), HIF-1α mRNA and protein levels were significantly upregulated in Caco-2 and HCT-116 cells, time-course analysis confirmed maximal HIF-1α stabilization at 48 hours, aligning with the experimental duration used for subsequent assays. Consistently, the mRNA expression levels of HIF-1α and HIF-1α-specific downstream target genes [including vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT1)] in CRC cells were significantly upregulated under hypoxic conditions (24hours and 48 hours). Similarly, co-immunoprecipitation experiments confirmed that TCF7L2 interacted with HIF-1α in Caco-2 and HCT116 cells exposed to hypoxia (Fig. 6 C). However, the physical interaction between HIF-1α and TCF7L2 could not be detected using the IP assay under normoxic conditions; this lack of interaction may be attributed to the low expression levels of HIF-1α in Caco-2 and HCT116 cells when exposed to normoxia (Fig. S1D). Based on the aforementioned results, the study assessed whether HIF-1α could directly bind to the TCF7L2 promoter, and regulate TCF7L2 gene transcription and expression. Using the online JASPAR platform, it was predicted that the HRE had a potential binding site on the TCF7L2 promoter region (Fig. 6 D). In addition, a ChIP assay was carried out, which demonstrated significant fold enrichment of HIF-1α binding to the HRE of the TCF7L2 promoter under hypoxia in Caco-2 and HCT116 cells, accompanied with a considerable decrease in enrichment after successful HIF-1α knockdown (Fig. 6 E). Similarly, under normoxic conditions, the binding state between HIF-1α and TCF7L2 could not be detected using the ChIP assay (Fig. S1D). Finally, to determine whether HIF-1α could independently influence the observed increase expression of TCF7L2 following exposure to hypoxia, the Caco-2 and HCT-116 cells were transfected with HIF-1α shRNA to specifically knock down HIF-1α expression. The results indicated that HIF-1α knockdown significantly abolished the hypoxia-induced increase in TCF7L2 expression at the protein and mRNA levels (Fig. 6 F and G). Taken together, these results demonstrated that hypoxia may induce upregulation of TCF7L2 in a HIF-1α-dependent manner. Upregulation of TCF7L2 and HIF-1α are associated with poor clinicopathological features in patients with CRC. To explore the clinical significance of TCF7L2 in CRC, the mRNA levels of TCF7L2 were measured in 104 pairs of CRC specimens and adjacent normal colorectal tissues. The results revealed that the expression levels of TCF7L2 in CRC specimens were significantly higher than those in the matched adjacent normal colorectal specimens (Fig. 7 A). The present study also quantified the mRNA expression levels of HIF-1α in the 104 pairs of CRC specimens and adjacent normal colorectal tissues. Similarly, the mRNA expression levels of HIF-1α were upregulated in CRC tissues (Fig. 7 B). Notably, a positive correlation was observed between HIF-1α expression and TCF7L2 expression in CRC specimens (Fig. 7 C). Consistently, elevated protein expression levels of HIF-1α and TCF7L2 were observed in 10 pairs of CRC tumor tissues than in adjacent normal colorectal tissues (Fig. 7 D). To further elucidate the role of TCF7L2 in both clinical phenotypes and prognostic outcomes of CRC, immunohistochemical (IHC) staining was performed on tissue specimens from 104 CRC patients. Based on TCF7L2 protein expression levels, patients were stratified into high- and low-expression groups. Statistical analysis revealed a significant positive correlation between TCF7L2 overexpression and advanced T stage as well as distant metastasis (Table I). Survival curve analysis further demonstrated that high TCF7L2 expression was associated with poorer overall survival (OS) rates (Fig. 7 E). Subsequently, patients were categorized into two subgroups according to the combined expression patterns of HIF-1α and TCF7L2: the HIF-1α High /TCF7L2 High group (n = 44) and the HIF-1α Low /TCF7L2 Low group (n = 26). Clinicopathological correlation analysis indicated that the dual-high expression subgroup exhibited more aggressive features, including higher T stage and increased metastatic potential (Table II). Survival analysis further confirmed that patients in the dual-high group had significantly worse prognosis compared to those in the dual-low group (Fig. 7 F), suggesting that co-detection of TCF7L2 and HIF-1α may serve as a prognostic biomarker panel for CRC. Representative IHC images depicting low and high expression patterns of both markers are shown in Supplementary Fig. S1E. Discussion The present study elucidated the biological function and underlying mechanism of TCF7L2 in CRC. The initial findings demonstrated that TCF7L2 upregulation under hypoxic conditions is implicated in cell proliferation, migration, invasion and EMT progression in CRC in vitro . In addition, TCF7L2 expression was associated with the maintenance of cancer stemness in CRC cells. Mechanistically, TCF7L2 was shown to promote CRC cell proliferation by activating the PI3K/AKT signaling pathway. Notably, TCF7L2 has been considered a key transcriptional regulator of HIF-1α, with HRE-binding sites located within the promoter region of HIF-1α, facilitating its transcriptional activation. The present in vivo studies revealed that TCF7L2 enhanced tumor growth in nude mice. Furthermore, TCF7L2 mRNA and protein expression levels were elevated in CRC tissues compared with those in adjacent normal tissues, and this upregulation was associated with aberrant clinical features. Moreover, patients with CRC exhibiting elevated expression levels of TCF7L2 and HIF-1α had a poorer prognosis than those with lower expression levels of these markers. Despite the development of ~ 22 novel HIF-1α inhibitors and the completion of numerous preclinical studies over the past decade ( 37 ), these efforts have yet to yield clinically viable therapies. Several challenges may impede clinical translation, including insufficient inhibitory potency, limited specificity, suboptimal pharmacokinetic profiles, toxicity concerns and flaws in clinical trial design. In future research, we aim to investigate the potential synergistic effects of co-administering TCF7L2 inhibitors with HIF-1α inhibitors to enhance their tumor-suppressive properties. The present study may offer valuable insights for the clinical translation of HIF-1 inhibitors. Numerous studies have reported that TCF7L2 is elevated in carcinoma tissues and is associated with poor prognosis ( 38 – 40 ). Previous studies have also demonstrated that the TCF7L2 protein is primarily localized in the cell nuclei of gastric cancer (GC) tissues and the cytoplasm of adjacent tissues ( 41 , 42 ). These findings suggested that TCF7L2 may exert a cancer-promoting role in the nucleus of GC cells and high TCF7L2 expression could be significantly associated with a poor prognosis in patients with GC. Functionally, TCF7L2 acts as a key transcriptional regulator of the urokinase-type plasminogen activator receptor (uPAR, encoded by the PLAUR gene), directly binding to specific regulatory elements within the PLAUR promoter region to modulate its transcription, which suggests that TCF7L2 may serve a vital role in regulating gastric cancer MKN45 cell proliferation, anoikis resistance and migration ( 41 ). Xiang et al ( 43 ) demonstrated that TCF7L2 can positively regulate aerobic glycolysis by suppressing EGLN2, leading to the upregulation of HIF-1α. This previous study also noted that TCF7L2 positively regulates HIF-1α stability and relevant glycolysis genes, such as GLUT1, hexokinase 2 and lactate dehydrogenase A in pancreatic cancer ( 43 ). Hypoxia is one of the most common and critical microenvironments in solid tumors. Various cellular responses to the hypoxic environment are regulated by a set of DNA-binding proteins, namely HIFs. As the predominant well-defined responsive regulator of hypoxic conditions in solid tumors, HIF-1α regulates multiple target genes through various biological pathways ( 44 ). Previous research has reported that HIF-1α functions as a negative regulator of human arrest defective 1 (hARD1)-mediated β-catenin acetylation, and β-catenin is deacetylated under hypoxic conditions due to its competition with HIF-1α for hARD1 binding. hARD1 is involved in the HIF-1α-mediated, hypoxic inactivation of TCF4 ( 45 ). The present ChIP analysis revealed that TCF7L2 is an important downstream targeting gene for HIF-1α. Inconsistent with the present findings, Kaidi et al ( 46 ) reported that HIF-1α interacts with β-catenin via its amine group (NH2) terminal domain, interfering with the β-catenin-TCF7L2 association, suggesting a complex regulatory network between HIF-1α and TCF7L2. EMT is a reversible process that was initially studied during embryo morphogenesis. In recent years, it has been shown that the state-switching between EMT and mesenchymal-epithelial transition serves a central role in various pathological processes, including tissue fibrosis, wound healing and the early stages of cancer development ( 47 ). Numerous studies have demonstrated that EMT is an early event of tumor metastasis ( 48 , 49 ). During EMT, cancer cells undergo phenotypic changes and epithelial cells morphologically transform into mesenchymal cells, resulting in enhanced cell motility and invasion. Epithelial cells have a typical apical-basal polarity structure, and the tight, adherent and gap junctions between these cells limit their ability to migrate and invade. During EMT activation, epithelial cells lose cell polarity and cell-cell junctions, and gain the ability to invade and migrate, transforming into cells with a mesenchymal morphology and characteristics ( 50 ). Given that the EMT and hypoxic microenvironment in tumors may share multiple signaling pathways, it has been suggested that hypoxia may induce EMT-like phenotypes in epithelial tumor cells ( 51 ). Among all of the signaling pathways involved in tumor hypoxia stimulation, the HIF-1α pathway is one of the most important pathways for hypoxia-induced EMT. Li et al ( 52 ) reported that hypoxia enhances migration, activating EMT and promoting MMP expression in hepatocellular cancer cells by targeting the AKT and HIF-1α/VEGF signaling pathways. Maugeri et al ( 53 ) reported that overexpression of pituitary adenylate cyclase-activating polypeptide is associated with hypoxia-induced EMT activation by regulating an important EMT transcription factor, Zinc finger E-box-binding homeobox-1, in glioblastoma ( 53 ). Shi et al ( 54 ) also demonstrated that PI3K/AKT signaling pathways are involved in hypoxia-induced EMT activation in CRC. The current study revealed that the mRNA and protein expression levels of HIF-1α and TCF7L2 were upregulated under the hypoxic tumor microenvironment in Caco-2 and HCT116 cell lines, which is in line with previous reports ( 32 , 55 ). Furthermore, the migration and invasion of CRC cells were markedly enhanced after hypoxia stimulation. Notably, the epithelial marker E-cadherin was downregulated, whereas the mesenchymal markers vimentin and N-cadherin, and the EMT transcription factors Snail and Slug were significantly increased under hypoxia. However, TCF7L2 knockdown abrogated hypoxia-induced EMT activation in CRC. Taken together, these findings indicated that TCF7L2 may be involved in the hypoxia-induced EMT progression of CRC. Accumulating evidence has shown that hypoxia has a notable role in the self-renewal and maintenance of stemness traits in CSCs and various carcinomas ( 56 , 57 ). CSCs have a high degree of metabolic adaptability and can survive in an oxygen-deficient environment. Additionally, the high acquisition and utilization of nutrients, such as glucose, enables CSCs to survive in restricted glucose level microenvironments, thereby promoting cell survival and tumorigenic potential ( 58 ). CSCs possess intrinsic stem cell characteristics, including self-renewal and resistance to chemotherapeutic agents. The capacity for tumor self-renewal is recognized as a defining hallmark of CSCs, contributing to tumor recurrence, drug resistance and metastasis. The sphere formation assay is a widely accepted technique for evaluating the formation of CSC-like cells. In this assay, single cells isolated from tumor spheres have demonstrated the ability to generate tumor spheres, thereby indicating the self-renewal capacity of cancer stem-like cells. The present study demonstrated that HCT-16 cells exhibited sphere-forming capability, which was sustained through the second passage under hypoxia, suggesting the important role of TCF7L2 in CRC stemness maintenance. Moreover, the results revealed that some typical stemness-associated genes, such as CD44, CD133, ALDH1A1, EpCAM, Nanog and OCT4, were significantly enriched in CRC cells after hypoxic stimulation. Conversely, TCF7L2 knockdown exhibited the opposite effects. Both CD44 and CD133 (also known as prominin-1) are putative stem markers for isolating CSCs from CRC ( 59 ). Therefore, the CD44 + /CD133 + subpopulation (defined as CRC CSCs) was isolated to further explore whether TCF7L2 was involved in hypoxia facilitating the development of CRC through enhanced stemness of CRC CSCs. The results demonstrated the independent role of TCF7L2 in cancer stemness maintenance of CRC. Previous research has demonstrated that β-catenin, a pivotal component of the Wnt signaling pathway, engages with HIF-1α across a range of physiological and pathological contexts, with the activation of the Wnt signaling pathway contributing to chemotherapy resistance ( 60 ). The present study identified an interaction between TCF7L2 and HIF-1α in CRC cells under hypoxic conditions. Given these findings, it is pertinent to investigate whether TCF7L2 facilitates HIF-1α in modulating the malignant behavior of CRC. Additionally, the PI3K/Akt pathway is integral to the regulation of metabolic processes, encompassing both matter and energy metabolism. The upregulation of HIF-1α expression and the enhancement of glycolysis in tumor cells are contingent upon the activation of the PI3K/Akt signaling pathway. Nevertheless, the precise mechanisms underlying this relationship remain inadequately understood. In light of these observations, our research aimed to investigate whether the PI3K/Akt pathway serves as a crucial mechanism through which TCF7L2 modulates the malignant behavior of CRC under hypoxic conditions. To date, the precise regulatory mechanism of TCF7L2 in CRC remains unclear. The present study identified a significant association between TCF7L2 expression and the activation of the PI3K/AKT signaling pathway. The PI3K/AKT signaling pathway is known to serve a crucial role in various biological processes, including cell proliferation, apoptosis and cell cycle progression. This pathway has also been reported to mediate the maintenance of stemness in various types of cancer, including liver cancer and CRC ( 61 , 62 ). The current study indicated that TCF7L2 may exert a proliferative effect on CRC cells by activating the PI3K/AKT signaling pathway. In conclusion, the findings of the present study demonstrated that TCF7L2 may serve a critical role in the progression of CRC. TCF7L2 upregulation was positively associated with poor clinical features in patients with CRC. Furthermore, to the best of our knowledge, the current study demonstrated previously unreported mechanistic crosstalk between HIF-1α and TCF7L2, indicating that TCF7L2 functions as a direct downstream target of HIF-1α in mediating tumor survival, metastasis and the maintenance of stemness properties in CRC. This study provides a theoretical basis for considering TCF7L2 and HIF-1α as potential therapeutic targets for CRC. Declarations Acknowledgements Not applicable. Funding No funding was received. Availability of data and materials The data generated in the present study may be requested from the corresponding author. Authors’ contributions KT, YC, JPG, and YL were instrumental in developing the study concepts and design. The literature review was conducted by YC, JPG, and YL. Experimental procedures were carried out by KT. Data analysis was performed by YC, JPG, and YL. The manuscript was written and revised collaboratively by KT, YC, JPG, and YL. The authenticity of all raw data has been verified by YC, JPG, and YL. All authors read and approved the final version of the manuscript. Ethics approval and consent to participate The present study was approved by the Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University (Chongqing, China, ethical approval no. 2023-326). Written informed consent was obtained from all participants or their parents. Patient consent for publication Written informed consent was obtained from patients or their families for publication of pathological sections or tissue sample images included in the study. Competing interests The authors declare that they have no competing interests. References Siegel RL, Giaquinto AN, Jemal A: Cancer statistics, 2024. CA Cancer J Clin. 74:12–49, 2024. Andrei P, Battuello P, Grasso G, Rovera E, Tesio N, Bardelli A: Integrated approaches for precision oncology in colorectal cancer: The more you know, the better. Semin Cancer Biol 84: 199–213, 2022. Valentini V, Coco C, Gambacorta MA, Barba MC, Meldolesi E: Evidence and research perspectives for surgeons in the European Rectal Cancer Consensus Conference (EURECA-CC2). Acta Chir Iugosl 57: 9–16, 2010. Siegel R, DeSantis C, Virgo K, Stein K, Mariotto A, Smith T, et al: Cancer treatment and survivorship statistics, 2012. CA Cancer J Clin 62: 220–241, 2012. Chen Z, Han F, Du Y, Shi H, Zhou W. Hypoxic microenvironment in cancer: molecular mechanisms and therapeutic interventions. Signal Transduct Target Ther 8: 70, 2023. Wicks, EE, Semenza, GL. Hypoxia-inducible factors: cancer progression and clinical translation. J Clin Invest 132: e159839, 2022. Schödel J, Grampp S, Maher ER, et al. Hypoxia, Hypoxia-inducible Transcription Factors, and Renal Cancer. Eur Urol. 69:646-657, 2016. Cowman SJ, Koh MY. Revisiting the HIF switch in the tumor and its immune microenvironment. Trends Cancer 8: 28-42, 2022. Schito L, Semenza GL: Hypoxia-Inducible Factors: Master Regulators of Cancer Progression. Trends cancer 2: 758–770, 2016. Semenza GL: Targeting HIF-1 for cancer therapy. Nat Rev Cancer 3: 721–732, 2003. de Heer EC, Jalving M, Harris AL. HIFs, angiogenesis, and metabolism: elusive enemies in breast cancer. J Clin Invest 130: 5074–5087, 2020. Wilson GK, Tennant DA, McKeating JA: Hypoxia inducible factors in liver disease and hepatocellular carcinoma: current understanding and future directions. J Hepatol 61:1397–1406, 2014. Lu T, Tang J, Shrestha B, Heath BR, Hong L, Lei YL, et al: Up-regulation of hypoxia-inducible factor antisense as a novel approach to treat ovarian cancer. Theranostics 10:6959–6976, 2020. Zeng L, Zhou HY, Tang NN, Zhang WF, He GJ, Hao B, et al: Wortmannin influences hypoxia-inducible factor-1 alpha expression and glycolysis in esophageal carcinoma cells. World J Gastroenterol 22: 4868–4880, 2016. Nagaraju GP, Bramhachari PV, Raghu G, El-Rayes BF: Hypoxia inducible factor-1α: Its role in colorectal carcinogenesis and metastasis. Cancer Lett 366: 11–18, 2015. Del Bosque-Plata L, Martínez-Martínez E, Espinoza-Camacho MÁ, Gragnoli C: The Role of TCF7L2 in Type 2 Diabetes. Diabetes 70: 1220–1228, 2021. Zhang T, Wang B, Su F, Gu B, Xiang L, Gao L, et al: TCF7L2 promotes anoikis resistance and metastasis of gastric cancer by transcriptionally activating PLAUR. Int J Biol Sci 18: 4560–4577, 2022. Ishiguro H, Wakasugi T, Terashita Y, Sakamoto N, Tanaka T, Sagawa H, et al: Nuclear expression of TCF4/TCF7L2 is correlated with poor prognosis in patients with esophageal squamous cell carcinoma. Cell Mol Biol Lett 21: 5, 2016. Rice SJ, Liu X, Hyland V, Liu Z, Belani CP: Mutations in genes connected with the TCF7L2 transcription factor are associated with a poor prognosis in non-small cell lung cancer. Lung Cancer 141: 97–100, 2020. Grant SFA. The TCF7L2 Locus: A Genetic Window Into the Pathogenesis of Type 1 and Type 2 Diabetes. Diabetes Care 42: 1624-1629, 2019. Chen CS, Huang CY, Huang SP, et al. Genetic interaction analysis of TCF7L2 for biochemical recurrence after radical prostatectomy in localized prostate cancer. Int J Med Sci 12: 243-247, 2015. Folsom AR, Pankow JS, Peacock JM, Bielinski SJ, Heiss G, Boerwinkle E: Variation in TCF7L2 and increased risk of colon cancer: the Atherosclerosis Risk in Communities (ARIC) Study. Diabetes Care 31: 905–909, 2008. Giles RH, Lolkema MP, Snijckers CM, Belderbos M, van der Groep P, Mans DA, et al: Interplay between VHL/HIF1alpha and Wnt/beta-catenin pathways during colorectal tumorigenesis. Oncogene 25: 3065–3070, 2006. Shi J, Lv Q, Miao D, Xiong Z, Wei Z, Wu S, et al: HIF2α Promotes Cancer Metastasis through TCF7L2-Dependent Fatty Acid Synthesis in ccRCC. Research (Washington, D.C.) 7: 0322, 2024. Chang J, Tian J, Yang Y, et al. A Rare Missense Variant in TCF7L2 Associates with Colorectal Cancer Risk by Interacting with a GWAS-Identified Regulatory Variant in the MYC Enhancer. Cancer Res 78: 5164-5172, 2018. Tang K, Toyozumi T, Murakami K, Sakata H, Kano M, Endo S, et al: HIF-1α stimulates the progression of oesophageal squamous cell carcinoma by activating the Wnt/β-catenin signalling pathway. Br J Cancer 127: 474–487, 2022. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-408,2001. Farina AR, Cappabianca L, Sebastiano M, Zelli V, Guadagni S, Mackay AR. Hypoxia-induced alternative splicing: the 11th Hallmark of Cancer. J Exp Clin Cancer Res. 39:110, 2020. Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell.141:52-67, 2010. Chen X, Zeh HJ, Kang R, Kroemer G, Tang D. Cell death in pancreatic cancer: from pathogenesis to therapy. Nat Rev Gastroenterol Hepatol. 18:804-823,2021. Duan S, Huang W, Liu X, et al. IMPDH2 promotes colorectal cancer progression through activation of the PI3K/AKT/mTOR and PI3K/AKT/FOXO1 signaling pathways. J Exp Clin Cancer Res. 37:304, 2018. Dong S, Liang S, Cheng Z, et al. ROS/PI3K/Akt and Wnt/β-catenin signalings activate HIF-1α-induced metabolic reprogramming to impart 5-fluorouracil resistance in colorectal cancer. J Exp Clin Cancer Res. 41:15, 2022. Chen Y, Xu X, Wang Y, et al. Hypoxia-induced SKA3 promoted cholangiocarcinoma progression and chemoresistance by enhancing fatty acid synthesis via the regulation of PAR-dependent HIF-1a deubiquitylation. J Exp Clin Cancer Res. 42:265, 2023. Zhang X, Yang L, Lei W, Hou Q, Huang M, Zhou R, et al: Single-cell sequencing reveals CD133+CD44--originating evolution and novel stemness related variants in human colorectal cancer. EBioMedicine 82: 104125, 2022. Koukourakis MI, Giatromanolaki A, Skarlatos J, et al. Hypoxia inducible factor (HIF-1a and HIF-2a) expression in early esophageal cancer and response to photodynamic therapy and radiotherapy. Cancer Res. 61:1830-1832, 2001. Boddy JL, Fox SB, Han C, et al. The androgen receptor is significantly associated with vascular endothelial growth factor and hypoxia sensing via hypoxia-inducible factors HIF-1a, HIF-2a, and the prolyl hydroxylases in human prostate cancer. Clin Cancer Res.11:7658-7663, 2005. Liu J, Gao Y, Zhang X. A patent review on hypoxia-inducible factor (HIF) modulators (2021-2023). Expert Opin Ther Pat.34:651-664, 2024. Del Bosque-Plata L, Hernández-Cortés EP, Gragnoli C. The broad pathogenetic role of TCF7L2 in human diseases beyond type 2 diabetes. J Cell Physiol 237: 301–312, 2022. Wu Q, Ma J, Wei J, Meng W, Wang Y, Shi M: lncRNA SNHG11 Promotes Gastric Cancer Progression by Activating the Wnt/β-Catenin Pathway and Oncogenic Autophagy. Mol Ther 29: 1258–1278, 2021. Wang D, Wu S, He J, Sun L, Zhu H, Zhang Y, et al: FAT4 overexpression promotes antitumor immunity by regulating the β-catenin/STT3/PD-L1 axis in cervical cancer. J Exp Clin Cancer Res 42: 222, 2023. Zhang T, Wang B, Su F, et al. TCF7L2 promotes anoikis resistance and metastasis of gastric cancer by transcriptionally activating PLAUR. Int J Biol Sci. 18:4560-4577, 2022. Ge L, Zhao G, Lan C, et al. MESP2 binds competitively to TCF4 to suppress gastric cancer progression by regulating the SKP2/p27 axis. Cell Death Discov. 9:79, 2023. Xiang J, Hu Q, Qin Y, Ji S, Xu W, Liu W, et al: TCF7L2 positively regulates aerobic glycolysis via the EGLN2/HIF-1α axis and indicates prognosis in pancreatic cancer. Cell Death Dis 9: 321, 2018. Yuan X, Ruan W, Bobrow B, Carmeliet P, Eltzschig HK: Targeting hypoxia-inducible factors: therapeutic opportunities and challenges. Nat Rev Drug Discov 23: 175–200, 2024. Lim JH, Chun YS, Park JW: Hypoxia-inducible factor-1alpha obstructs a Wnt signaling pathway by inhibiting the hARD1-mediated activation of beta-catenin. Cancer Res 68: 5177–5184, 2008. Kaidi A, Williams AC, Paraskeva C: Interaction between beta-catenin and HIF-1 promotes cellular adaptation to hypoxia. Nat Cell Biol 9: 210–217, 2007. Pastushenko I, Blanpain C: EMT Transition States during Tumor Progression and Metastasis. Trends Cell Biol 29: 212–226, 2019. Akhmetkaliyev A, Alibrahim N, Shafiee D, Tulchinsky E. EMT/MET plasticity in cancer and Go-or-Grow decisions in quiescence: the two sides of the same coin?. Mol Cancer. 22:90, 2023. Huang Y, Hong W, Wei X. The molecular mechanisms and therapeutic strategies of EMT in tumor progression and metastasis. J Hematol Oncol. 15:129, 2022. Davis FM, Stewart TA, Thompson EW, Monteith GR: Targeting EMT in cancer: opportunities for pharmacological intervention. Trends Pharmacol Sci 35: 479–488, 2014. Hapke RY, Haake SM: Hypoxia-induced epithelial to mesenchymal transition in cancer. Cancer Lett 487: 10–20, 2020. Li C, Wang Q, Shen S, Wei X, Li G: HIF-1α/VEGF signaling-mediated epithelial-mesenchymal transition and angiogenesis is critically involved in anti-metastasis effect of luteolin in melanoma cells. Phytother Res 33: 798–807, 2019. Maugeri G, D'Amico AG, Saccone S, Federico C, Rasà DM, Caltabiano R, et al: Effect of PACAP on Hypoxia-Induced Angiogenesis and Epithelial-Mesenchymal Transition in Glioblastoma. Biomedicines 9: 965, 2021. Shi Z, To SKY, Zhang S, Deng S, Artemenko M, Zhang M, et al. Hypoxia-induced Nur77 activates PI3K/Akt signaling via suppression of Dicer/let-7i-5p to induce epithelial-to-mesenchymal transition. Theranostics. 11:3376–3391, 2021. Zhang Q, Bai X, Chen W, et al. Wnt/β-catenin signaling enhances hypoxia-induced epithelial-mesenchymal transition in hepatocellular carcinoma via crosstalk with hif-1α signaling. Carcinogenesis. 34:962-973, 2013. Keith B, Simon MC. Hypoxia-inducible factors, stem cells, and cancer. Cell. 129:465-472, 2007. Zhang D, Yang L, Liu X, et al. Hypoxia modulates stem cell properties and induces EMT through N-glycosylation of EpCAM in breast cancer cells. J Cell Physiol. 235:3626-3633, 2020. Sun X, Lv X, Yan Y, Zhao Y, Ma R, He M, et al: Hypoxia-mediated cancer stem cell resistance and targeted therapy. Biomed Pharmacother 130: 110623, 2020. Wei F, Zhang T, Deng SC, Wei JC, Yang P, Wang Q, et al: PD-L1 promotes colorectal cancer stem cell expansion by activating HMGA1-dependent signaling pathways. Cancer Lett 450: 1–13, 2019. Boso D, Rampazzo E, Zanon C, et al. HIF-1α/Wnt signaling-dependent control of gene transcription regulates neuronal differentiation of glioblastoma stem cells. Theranostics. 2019;9(17):4860-4877. Stefani C, Miricescu D, Stanescu-Spinu II, et al. Growth Factors, PI3K/AKT/mTOR and MAPK Signaling Pathways in Colorectal Cancer Pathogenesis: Where Are We Now?. Int J Mol Sci. 22:10260, 2021. Tian LY, Smit DJ, Jücker M. The Role of PI3K/AKT/mTOR Signaling in Hepatocellular Carcinoma Metabolism. Int J Mol Sci.24:2652, 2023. Tables Table I. Association between TCF7L2 expression and the clinicopathological features of patients with colorectal cancer. TCF7L2 expression level Characteristic High Low P-value Sex 0.745 Male 36 24 Female 25 19 Age, years 0.644 >65 37 28 ≤65 24 15 T stage 0.027 a 1 1 3 2 9 15 3 41 22 4 10 3 N stage 0.781 N0 34 26 N1 17 12 N2 10 5 M stage 0.011 a M1 14 2 M0 47 41 Pathological stage 0.141 I 10 6 II 20 20 III 17 14 IV 14 3 a P<0.05. TCF7L2, transcription factor 7-like 2. Table II. Association among TCF7L2, expression HIF-1α expression and the clinicopathological features of patients with colorectal cancer. HIF-1α/TCF7L2 expression Characteristic HIF-1α High TCF7L2 High HIF-1α High TCF7L2 Low HIF-1α Low TCF7L2 High HIF-1α Low TCF7L2 Low P-value Sex 0.906 Male 27 9 9 15 Female 17 8 8 11 Age, years 0.435 >65 24 11 13 17 ≤65 20 6 4 9 T stage 0.020 a 1 1 0 0 3 2 6 3 3 12 3 30 13 11 9 4 7 1 3 2 N stage 0.776 N0 24 9 10 17 N1 14 5 3 7 N2 6 3 4 2 M stage 0.020 a M1 12 2 2 0 M0 32 15 15 26 Pathological stage 0.114 I 7 0 3 6 II 13 8 7 12 III 12 6 5 8 IV 12 3 2 0 a P<0.05. HIF, hypoxia-inducible factor; TCF7L2, transcription factor 7-like 2. Additional Declarations No competing interests reported. Supplementary Files Supplementaryfigure1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7002021","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":479918419,"identity":"beeb4d77-9ed8-4aec-a08d-d620ad30d8ad","order_by":0,"name":"KANG TANG","email":"","orcid":"","institution":"The Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"KANG","middleName":"","lastName":"TANG","suffix":""},{"id":479918420,"identity":"d2de664a-4b5e-4f33-b576-107b6e186352","order_by":1,"name":"YONG Cheng","email":"","orcid":"","institution":"The First Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"YONG","middleName":"","lastName":"Cheng","suffix":""},{"id":479918421,"identity":"4b017091-09f4-4a27-bd3e-c715ea88aa88","order_by":2,"name":"JIANPING GONG","email":"","orcid":"","institution":"The Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"JIANPING","middleName":"","lastName":"GONG","suffix":""},{"id":479918422,"identity":"f73684ff-e16c-4d62-a587-c6a50697bff8","order_by":3,"name":"YANG LI","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABUUlEQVRIie2RzUrDQBDHJywkHhJzbQi1r7Ah0CrW9lU2FPRUEYTicaWwXqpnC/oIQkAo9tawBy/Fc6CXSK4KES/VFnRT41dS9Cq4f5adndn9MTszAFJSf1Cr6M2umdphN3qg4phGovd7vYioGeJaPc6c/gIRIfITklkPhzvUNlIEfkM03XncewZi9QLqbl3VmzXNDBICjfLFKVGiewaVWv5jumv3T2BX1ELj9njbG3YRKhFouYOQIOecgTOkeQTbRg/2F1najBPMEQhk5AlEtQ0GBI8KWWYC8fzQo/YG402BoGmGaLPlSNXWpxmiMK74HKkfWdBSRO1sikaJJgfMOWaLWqrrBItaxrdd6+ym5PjfEdPklxN9no7yKI6emOiYGcRhctAoD65bQXLXqVdyWQBWMCjs5dPH2a7QdAGUipPRIoD5Fx9nVqH5l1JSUlL/Va+22nS8Xk2FVwAAAABJRU5ErkJggg==","orcid":"","institution":"The Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":true,"prefix":"","firstName":"YANG","middleName":"","lastName":"LI","suffix":""}],"badges":[],"createdAt":"2025-06-29 10:08:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7002021/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7002021/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85959616,"identity":"ffbc8368-f18e-4d63-9df6-b04c4d2541aa","added_by":"auto","created_at":"2025-07-03 15:25:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":300217,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of TCF7L2 in CRC cell lines. (A) RT-qPCR and (B) western blotting were used to detect the expression levels of TCF7L2 mRNA and protein in different CRC cell lines. HIEC-6 was used as a human normal small intestinal cell line and β-actin was used as a loading control (n=3). The mRNA and protein expression levels of TCF7L2 in Caco-2 and HCT-116 cells under N and H conditions were detected by (C) RT-qPCR and (D) western blotting (n=3). β-actin was used as a loading control. Data are presented as the mean ± SD. \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001, \u003csup\u003e****\u003c/sup\u003eP\u0026lt;0.0001. CRC, colorectal cancer; N, normoxia; H, hypoxia; RT-qPCR, reverse transcription-quantitative PCR; TCF7L2, transcription factor 7-like 2.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7002021/v1/321edaa69dd0cd5af5c9210e.png"},{"id":85958042,"identity":"9891d361-6e1f-4c06-b378-051faa539ab0","added_by":"auto","created_at":"2025-07-03 15:09:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":859866,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of TCF7L2 on cell survival, migration, invasion and EMT in CRC cell lines. (A) RT-qPCR and (B) western blotting were performed to confirm the knockdown efficiency of TCF7L2-shRNA in Caco-2 and HCT-116 cell lines (n=3). Evaluation of cell proliferation in the designated groups of (C) Caco-2 and (D) HCT-116 cells \u003cem\u003ein vitro\u003c/em\u003e using the Cell Counting Kit-8 assay (n=3). (E) Images of xenograft tumors in nude mice (n=5). (F) Growth curves of xenograft tumors from Caco-2 cells (n=5). (G) Growth curves of xenograft tumors from HCT-116 cells (n=5). (H) Measurement of glucose consumption levels in indicated CRC cells as determined by a glucose assay kit (n=3). (I) Representative images (Scar bar: 100 μm) and (J) statistical analysis of migration and invasion in Caco-2 and HCT-116 cells (n=3). (K) Protein expression levels of MMP2 and MMP9 were determined by western blotting (n=3). (L) RT-qPCR analysis of EMT markers in Caco-2 cells. (M) RT-qPCR analysis of EMT markers in HCT-116 cells. (N) Western blotting analysis of EMT markers in Caco-2 and HCT-116 cells (n=3). β-actin was used as loading control. Data are presented as the mean ± SD. \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001. CRC, colorectal cancer; EMT, epithelial mesenchymal transition; NC, negative control; ns, not significant; RT-qPCR, reverse transcription-quantitative PCR; sh, short hairpin; TCF7L2, transcription factor 7-like 2.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7002021/v1/a7f837a02a94910417359f8a.png"},{"id":85958758,"identity":"582cebcc-2169-43e9-ae6b-e827818296ed","added_by":"auto","created_at":"2025-07-03 15:17:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":620772,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of TCF7L2 on the apoptosis and cell cycle progression of CRC cell lines. (A) Representative plots and statistical analysis of cell apoptosis rate, as determined by FACS analysis using the FITC Annexin V Apoptosis Detection Kit (n=3). B Representative plots and statistical analysis of cell cycle progression in the indicated CRC cells, as analyzed by FACS analysis (n=3). (C) Protein expression levels of the cell cycle-related proteins cyclin D1 and PCNA were detected by western blotting (n=3). (D) Protein expression levels of PI3K, p-PI3K, Akt and p-Akt in the indicated cells were detected by western blot analysis (n=3). (E) Protein expression levels of TCF7L2, PI3K/AKT pathway activation and sensitivity to PI3K inhibition in the indicated CRC cells (n=3). β-actin was used as a loading control. (F) TCF7L2-overexpressing CRC cells were treated with the PI3K inhibitor LY294002, and cell proliferation was determined using the Cell Counting Kit-8 assay (n=3). Data are presented as the mean ± SD. \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001. CRC, colorectal cancer; FACS, fluorescence-activated cell sorting; LV, lentivirus; NC, negative control; ns, not significant; p-, phosphorylated; PCNA, proliferating cell nuclear antigen; sh, short hairpin; TCF7L2, transcription factor 7-like 2.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7002021/v1/9c3bc4170103915ecdcf2082.png"},{"id":85957936,"identity":"32d64fd3-2efc-4234-857b-675e450aef82","added_by":"auto","created_at":"2025-07-03 15:09:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":762186,"visible":true,"origin":"","legend":"\u003cp\u003eTCF7L2 participates in hypoxia-mediated chemoresistance. Cell viability was quantified using the Cell Counting Kit-8 assay in the indicated cells treated with (A) 5-FU or (C) LOHP for 72 h, respectively (N=3). Resistance to (B) 5-FU or (D) LOHP in the indicated cells, determined as the IC\u003csub\u003e50\u003c/sub\u003e value which was obtained from the inhibition curve (n=3). (E) 5-FU and LOHP induced apoptosis in the indicated cells, as determined by fluorescence-activated cell sorting analysis (n=3). Data are presented as the mean ± SD. \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01. 5-FU, 5-fluorouracil; LOHP, oxaliplatin; NC, negative control; ns, not significant; sh, short hairpin; TCF7L2, transcription factor 7-like 2.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7002021/v1/a5ac49db082d6ddfbc26490b.png"},{"id":85958043,"identity":"62fa2fb7-688b-4ae5-84ba-9f71105064cd","added_by":"auto","created_at":"2025-07-03 15:09:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":703938,"visible":true,"origin":"","legend":"\u003cp\u003eRole of TCF7L2 in cancer stemness maintenance of CRC cell lines. (A) Colony formation ability of CRC cells in the indicated groups was analyzed (n=3). (B) Sphere formation assay was used to assess the sphere formation abilities of the indicated CRC cell lines. (C) FACS analysis of the expression of cell-surface markers CD44 and CD133 in the indicated CRC cells (n=3). (D) mRNA and (E) protein expression levels of CSC-related markers in the indicated CRC cells were determined by RT-qPCR and western blotting (n=3). (F) Sphere formation ability of the indicated cell subpopulation was analyzed (n=3). (G) Representative flow cytometry (FACS) plots of CD44 and CD133 expression in CRC cell subpopulations (n=3). (H) Statistical quantification of CD44⁺ and CD133⁺ cell proportions in CRC cell subpopulations (n=3). Detection of the (I) mRNA and (J) protein expression levels of TCF7L2 and CSCs-related genes in the indicated cell subpopulations by RT-qPCR and western blotting. β-actin was used as a loading control (n=3). Data are presented as the mean ± SD. \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001. ALDH1A1, aldehyde dehydrogenase 1 family member A1; CRC, colorectal cancer; CSC, cancer stem cells; EpCAM, epithelial cell adhesion molecule; FACS, fluorescence-activated cell sorting; NC, negative control; ns, not significant; p-, phosphorylated; RT-qPCR, reverse transcription-quantitative PCR; sh, short hairpin; SOX2, SRY-box 2; TCF7L2, transcription factor 7-like 2.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7002021/v1/121b4ba6eadb48edaa4c0632.png"},{"id":85957695,"identity":"145f5d80-9f10-4998-bb1b-257d1c4fec2d","added_by":"auto","created_at":"2025-07-03 15:09:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":697866,"visible":true,"origin":"","legend":"\u003cp\u003eUpregulation of TCF7L2 expression in CRC cells in response to hypoxia depends on HIF-1α. (A) Relative mRNA expression levels of TCF7L2, HIF-1α, HIF-2α, VEGF and GLUT1 in Caco-2 and HCT-116 cells in response to different periods of hypoxia were quantitatively detected by RT-qPCR (n=3). (B) Protein expression levels of TCF7L2, HIF-1α, HIF-2α, VEGF and GLUT1 were determined by western blotting in Caco-2 and HCT-116 cells in response to different periods of hypoxia (n=3). (C) Endogenous interaction between HIF-1α and TCF7L2 in Caco-2 and HCT-116 cells under hypoxia was analyzed using a co-IP assay with anti-HIF-1α and anti-TCF7L2 antibodies. IgG was used as the negative control (n=3). (D) HIF-1α binding motif (upper panel); JASPAR online analysis predicted a potential HIF-1α binding HRE within the TCF7L2 gene promoter region, promoter region defined as 1.0-kb upstream of the transcriptional start site (lower panel). (E) Chromatin IP analysis showed that HIF-1α binds to the TCF7L2 promoter region (HRE), hypoxia enhances the binding of HIF-1α to the TCF7L2 promoter, and the binding is partially impaired by the knockdown of HIF-1α (N=3). (F) RT-qPCR and (G) western blotting demonstrated that hypoxia-induced mRNA and protein expression levels of TCF7L2 in Caco-2 and HCT-116 cells could be impaired by HIF-1α knockdown (n=3). β-actin was used as a loading control. Data are presented as the mean ± SD. \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001. CRC, colorectal cancer; GLUT1, glucose transporter 1; HIF, hypoxia-inducible factor; HRE, hypoxia response element; IB, immunoblot; IgG, immunoglobulin G; IP, immunoprecipitation; NC, negative control; ns, not significant; p-, phosphorylated; RT-qPCR, reverse transcription-quantitative PCR; sh, short hairpin; TCF7L2, transcription factor 7-like 2; VEGF, vascular endothelial growth factor.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7002021/v1/21c6e69e01d24796fdbf07fd.png"},{"id":85957848,"identity":"76e614e9-4613-4567-ad61-710409257d78","added_by":"auto","created_at":"2025-07-03 15:09:13","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":339641,"visible":true,"origin":"","legend":"\u003cp\u003eAberrantly elevated TCF7L2 and HIF-1α expression is associated with poor prognosis in patients with CRC. (A) TCF7L2 mRNA expression in 104 CRC tumor specimens and paired adjacent non-tumor tissues were analyzed by RT-qPCR (n=104). (B) HIF-1α mRNA expression in 104 CRC tumor specimens and paired adjacent non-tumor tissues were analyzed by RT-qPCR (n=104). (C) Correlation between the mRNA expression levels of TCF7L2 and HIF-1α in 104 CRC tumor tissues was assessed using Spearman correlation analysis (N=104; r\u003csub\u003es\u003c/sub\u003e=0.6077; P\u0026lt;0.001). (D) TCF7L2 and HIF-1α protein expression in 10 CRC tumor specimens and paired adjacent non-tumor tissues were analyzed by western blotting. β-actin was used as a loading control. (E) Kaplan-Meyer analysis of overall survival curves comparing patients with high (n=61) and low (n=43) TCF7L2 expression. (F) Kaplan-Meyer analysis of overall survival curves comparing HIF-1α\u003csup\u003eHigh\u003c/sup\u003e TCF7L2\u003csup\u003eHigh\u003c/sup\u003e (n=43) and HIF-1α\u003csup\u003eLow\u003c/sup\u003e TCF7L2\u003csup\u003eLow \u003c/sup\u003e(n=26) expressing patients. Data are presented as the mean ± SD. \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01. CRC, colorectal cancer; T: Tumor tissue, NT: Non-tumor tissue; HIF, hypoxia-inducible factor; RT-qPCR, reverse transcription-quantitative PCR; TCF7L2, transcription factor 7-like 2.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7002021/v1/8a6cc3bc82d0c775d990934a.png"},{"id":104808527,"identity":"0177aff9-b35a-4cd0-97f2-46e88a680b8f","added_by":"auto","created_at":"2026-03-17 12:38:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5350619,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7002021/v1/0630b50b-11c9-44ed-ac2b-ca8100625b47.pdf"},{"id":85957694,"identity":"0acaabca-1320-443d-b8a1-1fde126601b1","added_by":"auto","created_at":"2025-07-03 15:09:11","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6941798,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7002021/v1/d3b6155e9455b0a3a876b9a6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanistic insights into hypoxia-induced TCF7L2 upregulation and its oncogenic effects on colorectal cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer (CRC) is one of the most prevalent solid malignancies and the third leading cause of tumor-related mortality worldwide, with the third highest disease-specific death rate (1).\u003csup\u003e\u0026nbsp;\u003c/sup\u003eMultiple treatments, such as gene therapy, total mesorectal excision, neoadjuvant chemotherapy or chemoradiotherapy, and multidisciplinary team management, have been developed and widely applied in the clinic in recent decades (2). However, combination therapy, including targeted therapy for advanced CRC, has limited effectiveness, as patients with advanced CRC still exhibit poor survival, with a 5-year survival rate of ~12% (3,4). In addition, the molecular mechanisms underlying CRC initiation, progression and metastasis are yet to be fully elucidated. Therefore, identifying the precise biological mechanisms of CRC and exploring novel effective molecular therapeutic targets is imperative to improve survival in patients with CRC.\u003c/p\u003e\n\u003cp\u003eHypoxia is one of the most common and critical characteristics of the microenvironment during solid tumor progression\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(5). It has been reported that hypoxia-inducible factors (HIFs) participate in proliferation, inflammation, angiogenesis, invasion and distant metastasis in various types of solid carcinoma (6). Cancer cells undergo a series of complex adaptive changes in response to the hypoxic cellular microenvironment, including cell energy metabolism, proliferation, apoptosis, invasion and angiogenesis. HIF-1, a heterodimer consisting of \u0026alpha; and \u0026beta; subunits, is a primary regulator of cellular responses to hypoxia during tumor progression. Several studies have shown that HIF-1\u0026alpha; evades degradation under a hypoxic\u0026nbsp;microenvironment by inhibiting prolyl hydroxylase domain hydroxylation (7-8). HIF-1\u0026alpha; combines with the HIF-1\u0026beta; subunit to form a heterodimer and then translocates from the cytoplasm into the nucleus, where it interacts with specific DNA sequences containing hypoxia response elements (HREs), and mediates the transcription and expression of multiple downstream target genes\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(9). To date, \u0026gt;100 target genes downstream of HIF have been discovered, most of which are involved in the metastasis cascade, including epithelial-mesenchymal transition (EMT), extracellular matrix, enhanced tumor cell motility and angiogenesis\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(10). Clinical studies have demonstrated that elevated expression of HIF-1\u0026alpha; is positively associated with poor prognosis in most solid carcinoma types, including breast cancer (11), hepatocellular carcinoma (12), ovarian cancer\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(13), esophageal cancer\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(14) and CRC\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(15).\u003c/p\u003e\n\u003cp\u003eTranscription factor 7-like 2 (TCF7L2) directly regulates genes involved in metabolism and cell cycle control within adipocytes\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(16). Notably, TCF7L2 has been reported to be expressed in various types of non-mineralizing soft cancerous tissues, including in colon, esophageal, lung, skin and stomach cancer, suggesting that TCF7L2 may have an essential role in the carcinogenesis of malignant tumors (17-19). It has also been reported that the TCF7L2 gene is associated with type 2 diabetes (20) and is inversely associated with prostate cancer (21). Meanwhile, the association between TCF7L2 and colon cancer has been demonstrated in nondiabetic participants (22). Moreover, a previous study revealed that TCF7L2 may be upregulated in mammary epithelial cell-derived organoids and involved in EMT. Notably, abnormal cellular expression of the TCF7L2 protein has been shown to be positively associated with increased expression levels of HIF-1\u0026alpha; (23). Similarly, the hypoxic microenvironment can increase the expression of TCF7L2 in clear cell renal cell carcinoma (ccRCC) in a HIF-2\u0026alpha;-dependent manner, and hypoxia-regulated TCF7L2 high expression participates in ccRCC tumor survival and distant metastasis (24). However, few studies have explored the function of TCF7L2 in CRC (25).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study seeks to evaluate the biological functions, clinical relevance, and underlying mechanisms of TCF7L2 in CRC. The research specifically aims to elucidate the regulatory role of TCF7L2 in key cellular processes, including proliferation, apoptosis, EMT, and cancer stemness maintenance, under hypoxia and normoxia. Additionally, the study examines the clinical significance of TCF7L2 expression in CRC by analyzing its association with tumor stage, metastasis, and patient prognosis. Mechanistically, the investigation focuses on the HIF-1\u0026alpha;-dependent upregulation of TCF7L2, emphasizing HIF-1\u0026alpha;-mediated transcriptional activation and its interaction with the TCF7L2 promoter, as well as the subsequent downstream signaling pathway.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cem\u003eClinical specimens and cell culture.\u003c/em\u003e A total of 104 clinical CRC samples and adjacent non-tumor samples were obtained from the Department of Gastrointestinal Surgery, The Second Affiliated Hospital of Chongqing Medical University (Chongqing, China) between January 2009 and December 2013. No patients received preoperative radiotherapy or chemotherapy before surgery. All patients provided written informed consent. The inclusion criteria were as follows: i) A diagnosis of colorectal adenocarcinoma confirmed through pathological examination, with patients having undergone radical resection for CRC; ii) absence of preoperative chemoradiotherapy. The exclusion criteria were as follows: i) Preoperative pathological diagnosis of mucinous adenocarcinoma, signet ring cell carcinoma, adenosquamous carcinoma, medullary carcinoma, undifferentiated carcinoma or carcinosarcoma; ii) incomplete case records; iii) patients who underwent palliative surgery for CRC. The present study was approved by the Ethical Committee of The Second Affiliated Hospital of Chongqing Medical University (ethical approval no. 2023\u0026thinsp;\u0026minus;\u0026thinsp;326).\u003c/p\u003e \u003cp\u003eCRC cell lines Caco-2 (cat. no. HTB-37), HCT-116 (cat. no. CCL-247), HT-29 (cat. no. HTB-38), LoVo (cat. no. CCL-229), SW480 (cat. no. CCL-228) and SW620 (cat. no. CCL-227), and the human immortalized normal small intestine epithelium cell line HIEC-6 (cat. no. CRL-3266) were obtained from the American Type Culture Collection. All of the cell lines used in the present study were obtained from the Central Laboratory of Chongqing Medical University, which were purchased from ATCC. The cells were genotyped by short tandem repeat analysis and were tested for \u003cem\u003eMycoplasma\u003c/em\u003e before use. All CRC cell lines were cultured in Leibovitz\u0026rsquo;s L-15 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) in a 21% O\u003csub\u003e2\u003c/sub\u003e, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37˚C. HIEC-6 cells were cultured in Opti-MEM I Reduced Serum Medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 4% FBS and 10 ng/ml epidermal growth factor (EGF; MilliporeSigma) in a 21% O\u003csub\u003e2\u003c/sub\u003e, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37˚C. To mimic the hypoxic microenvironment, cells were cultured in a hypoxic cell incubator (Thermo Fisher Scientific, Inc.) supplemented with 1% O\u003csub\u003e2\u003c/sub\u003e, 5% CO\u003csub\u003e2\u003c/sub\u003e and 94% N\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eRNA interference.\u003c/em\u003e Transduction-ready TCF7L2 short hairpin RNA (shRNA) (h) Lentiviral Particles (cat. no. sc-43525-V) and HIF-1α shRNA (h) Lentiviral Particles (cat. no. sc-35561-V) (both from Santa Cruz Biotechnology, Inc.) were transduced into both Caco-2 and HCT-116 cells to establish stable knockdown of TCF7L2 or HIF-1α. Briefly, CRC cell lines (5x10\u003csup\u003e4\u003c/sup\u003e/well) were plated in a 6-well plate 24 h before lentiviral particle infection. The thawed lentiviral particles were then transduced into cells overnight with a multiplicity of infection of 4, alongside 5 \u0026micro;g/ml polybrene (Santa Cruz Biotechnology, Inc.). Stable clones expressing TCF7L2 or HIF-1α shRNA were selected and maintained using 10 \u0026micro;g/ml puromycin dihydrochloride (MilliporeSigma) for 14 days and harvest immediately for subsequent experiments. Control shRNA Lentiviral Particles (cat. no. sc-108080; Santa Cruz Biotechnology, Inc.) were transduced into cells as a negative control. In addition, TCF7L2 Lentiviral Activation Particles (h) (cat. no. sc-400607-LAC; Santa Cruz Biotechnology, Inc.) were transduced into Caco-2 and HCT-116 cells to establish stable overexpression of TCF7L2. Control Lentiviral Activation Particles (cat. no. sc-437282; Santa Cruz Biotechnology, Inc.) were transduced into cells as a negative control. The specific operation procedure and method were applied as previously described (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eRNA extraction and reverse transcription-quantitative PCR (RT-qPCR).\u003c/em\u003e Total RNA was isolated from tissues or cultured CRC cells using RNAiso plus (Takara Bio, Inc.) according to the manufacturer\u0026rsquo;s protocol. cDNA was then synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to manufacturer\u0026rsquo;s protocol. qPCR analyses were conducted by using \u003cem\u003eSsoFast EvaGreen Supermix\u003c/em\u003e (Bio-Rad) in Bio-Rad CFX96 system as previously described (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Thermocycling conditions included an initial enzyme activation at 95˚C for 30 sec, followed by 40 cycles of denaturation at 95˚C for 5 sec, annealing/extension at 60˚C for 5 sec. The relative expression levels of mRNA were calculated according to the 2\u003csup\u003e\u0026minus;ΔΔCq\u003c/sup\u003e method, where: ΔΔCq = Δ Cq (sample) - ΔCq (control) (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). The primer sequences used for qPCR analysis are listed in Table SI. All reactions were performed in triplicate.\u003c/p\u003e \u003cp\u003e\u003cem\u003eWestern blot analysis.\u003c/em\u003e Proteins were extracted from frozen tissues or cultured CRC cells using RIPA lysis buffer containing phosphatase inhibitor (MilliporeSigma). Subsequently, Protein concentrations were quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific) according to manufacturer\u0026rsquo;s protocol prior to loading\u0026thinsp;~\u0026thinsp;20 \u0026micro;g protein samples were separated electrophoresis on 10% by SDS-PAGE gels, transferred onto polyvinylidene fluoride membranes and blocked with 3% bovine serum albumin (MilliporeSigma) diluted with Tris-buffered saline containing 0.1% Tween 20 solution at room temperature for 1 h. The membranes were then incubated at 4˚C overnight with the primary antibodies. The information on all primary antibodies used in the present study is provided in Table SII. Subsequently, membranes were incubated at room temperature for 1 h with a horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) secondary antibody (1:2,000 dilution; cat. no. sc-2301; Santa Cruz Biotechnology) or HRP-conjugated anti-mouse IgG HRP secondary antibody (1:2,000 dilution; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.). Protein bands were visualized using the ECL Prime Western Blotting Detection Reagent (Amersham). Densitometric analysis of immunoblot bands was performed using Quantity One (Bio-Rad Quantity One version 4.6.2). The specific experimental methods were conducted as previously described (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eCell proliferation, colony formation and sphere formation assays.\u003c/em\u003e The cell proliferation assay was carried out using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Inc.) at indicated time points (6, 24, 48, 72 and 96 hours) according to the manufacturer\u0026rsquo;s protocol. Colony formation assays were performed as follows. Briefly, 1x10\u003csup\u003e3\u003c/sup\u003e cells/well were seeded into a 6-well plate and were cultured in complete cell medium for 12 days. Colonies in each group were then fixed and stained with a Diff-Quick kit (Sysmex Corporation), cells were fixed with the methanol-based solution (Component A ), at room temperature (RT) for 1 minute. Following fixation, colonies were stained sequentially with the Diff-Quick Stain Solution I (Component B, eosinophilic dye) at RT for 1 minute and Diff-Quick Stain Solution II (Component C, basophilic dye) at RT for 1 minute. A colony was defined as consisting of \u0026ge;\u0026thinsp;50 cells. Plating efficiency (PE) was calculated using the following formula: PE (%)\u0026thinsp;=\u0026thinsp;number of colonies formed/number of cells seeded x 100. For the sphere formation assay, 1x10\u003csup\u003e4\u003c/sup\u003e cells were cultured on low-adherence plates in 10 ml serum-free medium supplemented with EGF (10 ng/ml, Invitrogen), basic fibroblast growth factor (20 ng/ml, Invitrogen), 4% B27 (Invitrogen) and 4 \u0026micro;g/ml insulin (MilliporeSigma). A light microscope (Olympus Microscope) was used to visualize and quantify tumor spheres under bright-field conditions. Sphere formation efficiency (SFE) was computed using the following formula: SFE (%)\u0026thinsp;=\u0026thinsp;number of spheres formed at day 14/number of cells seeded x 100. The specific experimental methods were carried out as previously described (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eTranswell migration and invasion assays. In vitro\u003c/em\u003e migration (using Transwell plates that were not pre-coated with Matrigel) and invasion (using Transwell plates that were pre-coated with Matrigel at 37\u0026deg;C for 2 hours) assays were performed using 24-well Transwell plates (diameter, 6.5 mm; pore size, 8 \u0026micro;m; Corning, Inc.). Briefly, CRC cells (2x10\u003csup\u003e5\u003c/sup\u003e) were plated into the upper chamber was filled with serum-free medium, while the lower chamber contained complete medium supplemented with 10% fetal bovine serum (FBS) to drive cell migration and invasion. Transwell plates were incubated at 37˚C for 24 h (migration) or 30 h (invasion). Finally, migratory or invasive cells that were attached to the lower chamber were gently washed with PBS, fixed and stained with a Diff-Quick stain kit (Sysmex Corporation), the membrane was fixed with Component A for 1 minute at RT. Following fixation, the membrane was sequentially stained using the Solution I (eosin-based stain) for 1 minute at RT, followed by Solution II (methylene blue-based stain) for 1 minute at RT. Cellular staining was visualized using a bright field compound light microscope (Olympus). The specific experimental methods were conducted as previously described (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eCell apoptosis and cell cycle analyses.\u003c/em\u003e Cell apoptosis analysis was performed using the Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit (BD Biosciences) according to the manufacturer\u0026rsquo;s protocol. Briefly, CRC cells (5x10\u003csup\u003e5\u003c/sup\u003e cells) in each indicated group were harvested and washed three times in ice-cold PBS. Cells were then transferred into 500 \u0026micro;l Annexin V-binding buffer with 5 \u0026micro;l Annexin V-FITC and 5 \u0026micro;l (50 \u0026micro;g/ml) propidium iodide (PI), and were incubated at room temperature for 15 min in the dark. Flow cytometric analysis (BD FACSCalibur, BD Biosciences)\u003c/p\u003e \u003cp\u003ewas applied to quantify the percentage of apoptosis in CRC cells in each indicated group, and the data were analyzed with FlowJo software (version 10.7.2, BD Biosciences).\u003c/p\u003e \u003cp\u003eFor cell cycle analysis, 5x10\u003csup\u003e5\u003c/sup\u003e cells in each indicated group were harvested after treatment, washed with PBS, fixed in 70% ice-clod ethanol at 4˚C overnight, treated with RNaseA (50 \u0026micro;g/ml) and 0.1% Triton X-100, and stained with PI (40 \u0026micro;g/ml) at room temperature for 30 min in the dark, followed by fluorescence-activated cell sorting (FACS) analysis (BD FACSCalibur, BD Biosciences). To assess cell cycle distribution, DNA content was determined using ModFit LT 3.0 software (Verity Software House, Inc.). The specific experimental methods were performed as previously described (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e\u003cem\u003eXenograft mouse assay.\u003c/em\u003e A total of 20 4-6-week-old male BALB/c-nu/nu mice (weighing 18\u0026ndash;20 g each) were purchased from the Laboratory Animal Center of Chongqing Medical University and were used for xenograft tumorigenicity assays. Mice were randomly allocated to each indicated group (n\u0026thinsp;=\u0026thinsp;5/group) and were housed in specific pathogen-free facilities under standard housing conditions with a 12-h light/dark cycle. Mice were provided ad libitum access to food and water, with housing conditions maintained at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 50% \u0026plusmn; 5% humidity. The animal experiment was designed to be a single-blind trial. Briefly, a final concentration of 2x10\u003csup\u003e6\u003c/sup\u003e Caco-2 or HCT-116 cells in each group was suspended in 200 \u0026micro;l medium with Matrigel (1:1) and subcutaneously injected into the right rear flank of nude mice. Mice were randomly divided into four groups as follows: Caco-2 Nc-shRNA (Caco-2 cells transfected with negative control shRNA), Caco-2 TFC7L2-shRNA (Caco-2 cells transfected with TCF7L2 shRNA), HCT-116 Nc-shRNA (HCT-116 cells transfected with negative control shRNA), HCT-116 TFC7L2-shRNA (HCT-116 cells transfected with TCF7L2 shRNA). Tumor size was measured twice weekly using calipers. Mice were sacrificed 6 weeks after injection by cervical dislocation under ether anesthesia. Humane endpoints were predefined as tumor volume exceeding 2000 mm\u0026sup3;. The tumor volume was calculated using the following formula: Tumor volume (mm\u003csup\u003e3\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;0.5 x [length (mm)] x [width (mm)\u003csup\u003e2\u003c/sup\u003e]. All animal experiments were approved by the Animal Ethics Committee of Chongqing Medical University (ethical approval no. 2023\u0026thinsp;\u0026minus;\u0026thinsp;326).\u003c/p\u003e \u003cp\u003e \u003cem\u003eGlucose assay.\u003c/em\u003e The glucose concentration in the culture supernatant was determined using the Glucose Assay Kit-WST (Dojindo Laboratories, Inc.). Briefly, prior to the experiment, standard curves were drawn with a standard glucose solution (0-0.5 \u0026micro;M). Cells (5x10\u003csup\u003e5\u003c/sup\u003e cells/well) were seeded in a 6-well microplate and were cultured at 37˚C for 24 h. Subsequently, 100 \u0026micro;l supernatant was transferred to a 1.5-ml microtube and diluted 40-fold with double distilled water to generate the sample solution. Approximately 50 \u0026micro;l sample solution and 50 \u0026micro;l working solution were then added to a 96-well microplate, which was incubated at 37˚C for 30 min. The absorbance of each well was measured at 450 nm using a microplate reader and glucose concentration in each sample solution was calculated using the aforementioned standard curve.\u003c/p\u003e \u003cp\u003e \u003cem\u003eFlow cytometry and magnetic cell sorting.\u003c/em\u003e Briefly, ~5x10\u003csup\u003e5\u003c/sup\u003e CRC cells in each indicated group were suspended in 200 \u0026micro;l cell staining buffer (cat. no. 420201; BioLegend, Inc.) and labeled with 10 \u0026micro;l FITC-conjugated anti-human CD44 (cat. no. 338804; BioLegend, Inc.) or phycoerythrin (PE)-conjugated anti-human CD133 (cat. no. 372804; BioLegend, Inc.) at 4˚C for 15 min in the dark. Samples were then centrifuged at 350 x g for 5 min at 4˚C and washed twice with 2 ml cell staining buffer. Subsequently, CD44 or CD133 expression was examined by flow cytometry (BD FACSCalibur, BD Biosciences), the data were analyzed with FlowJo software (version 10.7.2, BD Biosciences). FACS of CD44\u003csup\u003e+\u003c/sup\u003e/CD133\u003csup\u003e+\u003c/sup\u003e double-positive cells and CD44\u003csup\u003e\u0026minus;\u003c/sup\u003e/CD133\u003csup\u003e\u0026minus;\u003c/sup\u003e double-negative HCT-116 cells was performed using a FACS ARIA II high-speed cell sorter (BD Biosciences) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003e \u003cem\u003eChemical and chemotherapy drug treatment.\u003c/em\u003e The PI3K inhibitor LY294002 (MilliporeSigma) was added to the cell culture medium at 37\u0026deg;C and a final concentration of 10 \u0026micro;M 24 h prior to the start of experiments assessing TCF7L2-mediated cell proliferation and PI3K/AKT signaling activation. The working concentration and processing time of LY294002 were selected based on the manufacturer\u0026rsquo;s protocol. The chemoresistance of CRC cells to 5-fluorouracil (5-FU) or oxaliplatin (LOHP) was analyzed using the CCK-8. The concentration gradient of 5-FU and LOHP was set according to a previous publication (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Briefly, 5x10\u003csup\u003e3\u003c/sup\u003e cells (per well) in each indicated group (Normoxia, hypoxia, hypoxia Negative Control (NC)-sh, hypoxia TCF7L2-sh) were seeded into a 96-well plate and incubated with different concentrations (0, 1, 2, 4, 6, 8,10, 50 and 100 \u0026micro;g/ml) of 5-FU or LOHP for 72 h at 37˚C. Subsequently, the medium was replaced with fresh medium containing 100 \u0026micro;l CCK-8 solution. Optical density values were determined at 450 nm using a microplate reader (Sanyo). Drug response curves and half-maximal inhibitory concentration (IC₅₀) values were generated using GraphPad Prism 8 software (GraphPad Software) via nonlinear regression analysis of dose\u0026ndash;response data.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCo-immunoprecipitation assay.\u003c/em\u003e Co-immunoprecipitation was carried out using the Dynabeads Protein G Immunoprecipitation Kit (cat. no. 10004D, Thermo Fisher Scientific, Inc.), according to the manufacturer\u0026rsquo;s instructions. Antibody-conjugated magnetic beads were generated by coupling 5 \u0026micro;g anti-HIF-1α (1:150 dilution; cat. no. Ab1; Abcam) or anti-TCF7L2 (1:30 dilution; cat. no. sc-166699; Santa Cruz Biotechnology, Inc.) antibodies with 50 \u0026micro;l (1.5 mg) magnetic beads, followed by resuspension in 200 \u0026micro;l antibody binding and elution buffer, and incubation with gentle rotation at room temperature for 15 min. Following incubation, 500 \u0026micro;l cell lysates containing the antigen were gently pipetted to resuspend the pre-prepared magnetic bead-antibody complex. Cell lysates were further incubated with rotation at 4˚C overnight to allow the antigen to bind to the magnetic bead-antibody complex. Finally, the magnetic bead-antibody-antigen complex was eluted in 20 \u0026micro;l elution buffer, and 20 \u0026micro;l eluted samples were mixed with 20 \u0026micro;l protein loading buffer. The mixture was heated at 95˚C for 5 min for SDS-PAGE and western blot analysis as aforementioned. Normal mouse IgG (1:30 dilution; cat. no. CST-7076S; Cell Signaling Technology, Inc.) was used as a negative control antibody. The specific experimental methods were conducted as previously described (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eChromatin immunoprecipitation (ChIP) assay.\u003c/em\u003e The promoter region of TCF7L2 was identified using publicly available online resources (NC_000010.11:c112950191-112950182, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/nuccore/NC_000010.11?from=112950182\u0026amp;to=112950191\u0026amp;strand=true\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/nuccore/NC_000010.11?from=112950182\u0026amp;to=112950191\u0026amp;strand=true\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The analysis concentrated on the first 1,000 base pairs upstream of the transcription start site. Utilizing computational screening with algorithms from the JASPAR database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://jaspar.genereg.net/\u003c/span\u003e\u003cspan address=\"http://jaspar.genereg.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), HREs or HIF-1 binding sites within the promoter region were identified, specifically within the 1,000 base pairs upstream of the TCF7L2 transcription start site (TSS). The ChIP assay was performed using the Simple ChIP Plus Enzymatic Chromatin IP Kit (Agarose Beads; cat. no. CST-9005; Cell Signaling Technology, Inc.) according to manufacturer\u0026rsquo;s protocol. Briefly, ~\u0026thinsp;5 \u0026micro;g anti-HIF-1α (1:150 dilution; cat. no. Ab1; Abcam) or negative control mouse IgG antibodies (1:30 dilution; cat. no. CST-7076S; Cell Signaling Technology, Inc.) were used for immunoprecipitation in ChIP reactions. Quantitative real-time PCR (qPCR) was performed immediately following the ChIP assay. The primers designed for ChIP assays are listed in Table SI. The specific experimental methods were performed as previously described (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eImmunohistochemistry (IHC).\u003c/em\u003e IHC was conducted on paraformaldehyde-fixed, paraffin-embedded CRC patients tumor tissue sections. Tumor tissues were fixed in 4% paraformaldehyde at room temperature for 24 hours, embedded in paraffin, and serially sectioned into 4-\u0026micro;m-thick slices. Sections were dewaxed with xylene and rehydrated through a gradient ethanol series (100%, 95%, 85%, 70%, v/v). Endogenous peroxidase activity was quenched by incubation with 3% hydrogen peroxide (H₂O₂) for 10 minutes at room temperature. Antigen retrieval was performed via microwave heating in 0.01 mol/L sodium citrate buffer (pH 6.0) at 95\u0026deg;C for 15 minutes, followed by cooling to room temperature. Sections were blocked with 5% bovine serum albumin (BSA; MilliporeSigma) in phosphate-buffered saline (PBS) for 20 minutes at room temperature to reduce nonspecific binding. Primary antibodies against HIF-1α (1:100 dilution; cat. no. ab1, Abcam) or TCF7L2 (1:50 dilution; cat. no. sc166699, Santa Cruz Biotechnology, Inc.) were applied and incubated overnight at 4\u0026deg;C in a humidified chamber. After washing with PBS, sections were probed with horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody (1:2,000 dilution; cat. no. sc-2005, Santa Cruz Biotechnology, Inc.) for 20 minutes at room temperature. Color development was achieved using 3,3'-diaminobenzidine (DAB; cat. no. 3468, Dako) according to the manufacturer\u0026rsquo;s instructions. Slides were counterstained with hematoxylin, dehydrated, cleared, and coverslipped. Staining was visualized and imaged under a bright-field light microscope (Olympus). Quantification of positive cell percentages was performed using ImageJ 1.48 software (National Institutes of Health), with mean values calculated from three independent fields per section. Protein expression levels were assessed independently by two investigators in collaboration with a pathologist, all of whom were blinded to the clinical information of the patients. The methodology for evaluating IHC staining results was structured as follows: The intensity score was categorized as 0 for no staining, 1 for weak staining, 2 for moderate staining and 3 for strong staining. The percentage score was defined as Score 0 for staining covering 0\u0026ndash;4% positive cells, Score 1 for 5\u0026ndash;24% positive cells, Score 2 for 25\u0026ndash;49% positive cells, Score 3 for 50\u0026ndash;74% positive cells, and Score 4 for 75\u0026ndash;100% positive cells. The final IHC score was calculated by multiplying the intensity score by the percentage score. High expression levels for HIF-1α and TCF4/TCF7L2 were identified as having final IHC scores of 1\u0026thinsp;+\u0026thinsp;and 3+, respectively.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStatistical analysis.\u003c/em\u003e Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from three independent experimental repeats. All statistical analyses were performed using GraphPad Prism 8.0 (Dotmatics) and SPSS version 22.0 (IBM Corp.). The unpaired Student\u0026rsquo;s t-test was used for comparisons between two independent groups, whereas a paired two-tailed Student\u0026rsquo;s t-test was used to compare expression between CRC samples and adjacent non-tumor tissues. One-way analysis of variance was applied for multiple group comparisons followed by Tukey's multiple comparison test. Correlation analyses between groups were assessed using Spearman rank correlation. Categorical variables were assessed using the χ\u003csup\u003e2\u003c/sup\u003e test or Fisher\u0026rsquo;s exact test. The Kaplan-Meier log-rank test was used to analyze the overall survival curve data. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate a statistically significant difference.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003eTCF7L2 expression is upregulated in CRC cell lines under hypoxia.\u003c/em\u003e TCF7L2 mRNA and protein expression levels were first evaluated in Caco-2, HCT-116, HT-29, LoVo, SW480 and SW620 cells. HIEC-6 cells were selected as a normal small intestine epithelial cell line. The results showed that the mRNA levels of TCF7L2 were markedly upregulated in CRC cell lines compared with those in the HIEC-6 cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Western blot analysis revealed a similar trend in TCF7L2 protein expression levels in CRC cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Hypoxia is one of the most critical niches during solid tumor progression (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Subsequently, the mRNA and protein levels of TCF7L2 were revealed to be significantly upregulated in Caco-2 and HCT-116 cells under hypoxic conditions compared with under normoxia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and D). Given that TCF7L2 was significantly upregulated in these two cell lines under hypoxic conditions, these two cell lines were selected for subsequent experiments to evaluate the biological impacts and investigate the mechanisms of TCF7L2 knockdown in colorectal cancer cells under hypoxia.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTCF7L2 promotes CRC cell proliferation, migration, invasion and EMT in the presence of hypoxia.\u003c/em\u003e Subsequently, stable TCF7L2 knockdown was induced in Caco-2 and HCT-116 cell lines using shRNA lentiviral particles to further elucidate the biological roles of TCF7L2 in CRC cell lines. RT-qPCR and western blot analysis were performed post-transduction with TCF7L2-shRNA to examine the mRNA and protein expression levels of TCF7L2, respectively. The results showed that cells with TCF7L2 knockdown had significantly lower levels of TCF7L2 mRNA and protein than negative control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B). The role of TCF7L2 in Caco-2 and HCT-116 cell proliferation was determined \u003cem\u003ein vitro\u003c/em\u003e using a CCK-8 assay. The results revealed that the proliferation rates of Caco-2 and HCT-116 cells were significantly increased under hypoxic conditions; however, TCF7L2 knockdown markedly inhibited hypoxia-induced hyper-proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D). Furthermore, it was observed that the proliferative capacity of HCT-116 and Caco-2 cells was markedly reduced following the knockdown of TCF7L2 under normoxic conditions (Fig. S1A and B). Given that TCF7L2 serves an important role in CRC cell proliferation \u003cem\u003ein vitro\u003c/em\u003e, the impact of TCF7L2 on the tumorigenicity of CRC cells \u003cem\u003ein vivo\u003c/em\u003e was investigated. The results revealed that TCF7L2 knockdown significantly suppressed tumor growth \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Taken together, these results indicates that TCF7L2 may have a vital role in cancer progression both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eGlucose is essential for cell proliferation, growth and survival. Thus, the Glucose Assay Kit-WST, which enables the quantitation of glucose as a substrate in energy metabolism, was utilized to assess the effects of TCF7L2 on cancer metabolism. It was revealed that the levels of glucose were decreased in the Caco-2 and HCT-116 cell supernatant after hypoxic stimulation; however, TCF7L2 knockdown reversed this trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH), thus suggesting that TCF7L2 may affect the efficiency of glucose utilization in CRC cells.\u003c/p\u003e \u003cp\u003eMetastasis is considered a crucial event during CRC development. Subsequently, Transwell migration and invasion assays were employed to investigate the role of TCF7L2 in hypoxia-induced CRC metastasis capability. The results showed that Caco-2 and HCT-116 cells exhibited significantly enhanced cell migration and invasion under hypoxic conditions; however, TCF7L2 knockdown blocked Caco-2 and HCT-116 cell migration and invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI and J). MMPs have been shown to serve an important role in tumor invasion and metastasis(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), and MMP2 and MMP9 are critical MMPs that regulate cell migration and invasion. In the present study, MMP9, but not MMP2, was highly expressed in Caco-2 and HCT-116 cells under hypoxic conditions; however, TCF7L2 knockdown decreased the expression levels of MMP9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK).\u003c/p\u003e \u003cp\u003eAccumulating evidence has confirmed that hypoxia has an important role in cancer metastasis and EMT. To further explore the molecular mechanism underlying the role of TCF7L2 in CRC development, the expression level of EMT markers and related transcription factors were detected. The results showed that hypoxia significantly downregulated the epithelial marker E-cadherin, but upregulated the mRNA expression levels of the mesenchymal markers N-cadherin and vimentin, as well as transcription factors Snail and Slug (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL and M). Conversely, TCF7L2 knockdown showed the opposite expression pattern of EMT-related genes in Caco-2 and HCT116 cells under hypoxic conditions. Consistent with the mRNA expression profiles, western blot analysis confirmed that hypoxia significantly promoted EMT progression in CRC cells; however, TCF7L2 knockdown partially reversed the role of hypoxia in EMT activation in Caco-2 and HCT116 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eN). Collectively, these results indicated that TCF7L2 may stimulate the survival and metastatic ability of CRC cell lines.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTCF7L2 is involved in hypoxia-induced apoptosis resistance and cell cycle arrest in CRC cell lines.\u003c/em\u003e Apoptosis resistance serves an essential role during the response of tumor progression to hypoxia (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Therefore, the apoptosis rate was detected to further investigate the role of TCF7L2 in Caco-2 and HCT-116 cells under hypoxic conditions. The results showed that the apoptosis rate was significantly decreased in Caco-2 and HCT-116 cells under hypoxic conditions; however, shRNA-mediated TCF7L2 knockdown significantly reduced hypoxia-induced apoptosis resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eFurthermore, cell cycle progression in Caco-2 and HCT-116 cells was examined by analyzing the distribution of cell cycle phases to further assess the role of TCF7L2 in the survival of CRC cells. The results revealed that the proportion of Caco-2 and HCT-116 cells in the G\u003csub\u003e0\u003c/sub\u003e/G\u003csub\u003e1\u003c/sub\u003e phase was significantly decreased under hypoxic conditions; however, TCF7L2 knockdown increased the proportion of cells in the G\u003csub\u003e0\u003c/sub\u003e/G\u003csub\u003e1\u003c/sub\u003e phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In addition, the protein expression levels of cyclin D1 and proliferating cell nuclear antigen (PCNA) were significantly increased in Caco-2 and HCT-116 cells under hypoxic conditions; conversely, TCF7L2 knockdown downregulated the expression levels of cyclin D1 and PCNA in these cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Taken together, these data suggested that TCF7L2 may promote CRC cell survival by inducing apoptosis resistance and cell cycle G\u003csub\u003e1\u003c/sub\u003e/S transition.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTCF7L2 promotes CRC cell proliferation via the PI3K/Akt signaling pathway.\u003c/em\u003e Based on the aforementioned findings that the PI3K/Akt signaling pathway regulates the proliferation of CRC cells (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), the current study further explored the protein expression levels of p-PI3K p85 and p-AKT1 (Ser 473) in Caco-2 and HCT116 cells exposed to hypoxia. The results demonstrated that these proteins were elevated under hypoxia, whereas TCF7L2 knockdown reduced the hypoxia-induced changes to these protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). To further confirm whether the PI3K/Akt signaling pathway participates in TCF7L2-induced cell proliferation, Caco-2 and HCT116 cells with successfully stable TCF7L2 overexpression were pretreated with the PI3K inhibitor LY294002 (10 \u0026micro;M) for 24 h to block the PI3K/Akt signaling pathway. It was observed that LY294002 treatment almost completely blocked the expression levels of p-PI3K p85 and p-AKT1 (Ser 473) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Moreover, the enhanced cell proliferation detected following TCF7L2 overexpression was partially reversed by the inhibition of PI3K in CRC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Collectively, these findings indicated that the PI3K/Akt signaling pathway may have a crucial role in TCF7L2-mediated proliferation of CRC cells.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTCF7L2 is involved in hypoxia-induced chemoresistance in CRC cell lines.\u003c/em\u003e Studies have demonstrated that hypoxia influences the occurrence of chemoresistance in various types of solid tumors due to the activation of HIF-1α signaling (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). To investigate whether TCF7L2 contributed to the development of chemoresistance of CRC cells exposed to hypoxia \u003cem\u003ein vitro\u003c/em\u003e, Caco-2 and HCT116 cells were treated with difference concentrations of 5-FU or LOHP, followed by calculation of the cell survival rate and IC\u003csub\u003e50\u003c/sub\u003e. To explore drug-induced apoptosis, Caco-2 and HCT116 cells were pretreated with 5 \u0026micro;g/ml 5-FU or LOHP for 72 h. The results showed that 5-FU or LOHP induced a dose-dependent decrease in the survival rate of Caco-2 and HCT116 cells. As expected, hypoxia stimulation triggered enhanced resistance of Caco-2 and HCT116 cells to 5-FU and LOHP intervention, with the IC\u003csub\u003e50\u003c/sub\u003e values of CRC cells exposed to hypoxia found to be significantly higher compared with those exposed to normoxia. Conversely, TCF7L2 knockdown in Caco-2 and HCT116 cells abrogated the hypoxia-induced chemoresistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D). Further analysis revealed that hypoxia caused a significant reduction in the drug-induced apoptosis of CRC cells, whereas TCF7L2 knockdown abolished this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). These findings indicated that TCF7L2 was involved in the regulation of chemosensitivity in CRC cells.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTCF7L2 contributes to the maintenance of cancer stem cell (CSC)-like phenotypes of CRC cells.\u003c/em\u003e Given the potential role of TCF7L2 in enhancing chemoresistance in CRC cells, the current study subsequently explored whether TCF7L2 may regulate chemoresistance of CRC cells. Specifically, colony formation and sphere formation assays were conducted on Caco-2 and HCT116 cells exposed to hypoxia. The results indicated that hypoxia promoted the clonogenic ability of Caco-2 and HCT116 cells, whereas TCF7L2 knockdown induced the opposite effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Regarding tumor sphere formation, Caco-2 cells were unable to form spheres in low-adherent, serum-free and growth factor medium compared with the HCT116 cells (Fig. S1C). The results of the assay indicated that hypoxia caused significant sphere formation of HCT-116 cells, with similar findings observed in the secondary passage; conversely, TCF7L2 knockdown inhibited sphere formation and the propagation of HCT-116 cells cultured in hypoxic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Taken together, these results indicated that TCF7L2 participated in the regulatory effects of hypoxia on the CSC properties of CRC cells. To verify these observations, the expression of some common stemness-related markers were investigated by FACS (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), RT-qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) and western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE) in HCT116 cells. The results indicated that the expression of stemness-related markers was significantly elevated under hypoxia; however, TCF7L2 knockdown in HCT-116 cells decreased the expression levels of cancer stemness markers, including CD44, CD133, epithelial cell adhesion molecule (EpCAM), and Nanog. Notably, no significant difference was observed for aldehyde dehydrogenase 1 family member A1 (ALDH1A1) or OCT3/4. Taken together, these findings demonstrated that TCF7L2 was involved in the regulatory effects of hypoxia on the stemness properties of CRC cells.\u003c/p\u003e \u003cp\u003eNext, the present study aimed to clarify whether TCF7L2 could independently affect the stemness of CRC cells. It has been reported that cellular surface proteins CD44 and CD133 are potential markers for specific subpopulations of HCT-116 cells (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). TCF7L2 knockdown was first established in bulk HCT-116 cells via lentiviral shRNA transduction. After selecting for stable knockdown, cells were labeled with CD44 and CD133 antibodies, and the CD44\u003csup\u003e+\u003c/sup\u003e/CD133\u003csup\u003e+\u003c/sup\u003e HCT-116 subpopulation was purified by FACS. The results of the present study showed that CD44\u003csup\u003e+\u003c/sup\u003e/CD133\u003csup\u003e+\u003c/sup\u003e HCT-116 subpopulations displayed enhanced SFE, whereas HCT-116 CD44\u003csup\u003e\u0026minus;\u003c/sup\u003e/CD133\u003csup\u003e\u0026minus;\u003c/sup\u003e subpopulations formed fewer spheres; as expected, TCF7L2 knockdown in HCT-116 CD44\u003csup\u003e+\u003c/sup\u003e/CD133\u003csup\u003e+\u003c/sup\u003e subpopulations induced a significant inhibition in SFE (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Next, flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and H), RT-qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI) and western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ) were performed to further determine the effect of TCF7L2 on the expression of CSC markers in regulating cancer stemness. The expression levels of CD44, CD133, ALDH1A1, SRY-box 2 (SOX2), EpCAM, Nanog and OCT4, which are widely accepted biomarkers of stemness for most solid tumors, were analyzed. The findings indicated that, apart from SOX2, other stemness markers such as CD44, CD133, ALDH1A1, EpCAM, Nanog and OCT3/4 were markedly upregulated in CD44\u003csup\u003e+\u003c/sup\u003e/CD133\u003csup\u003e+\u003c/sup\u003e HCT-116 cells. Conversely, these markers exhibited a significant downregulation following the knockdown of TCF7L2. Collectively, these findings suggested that TCF7L2 could independently regulate the stemness of CRC cells.\u003c/p\u003e \u003cp\u003e \u003cem\u003eHypoxia induces the upregulation of TCF7L2 via direct transcriptional stimulation of HIF-1α.\u003c/em\u003e Hypoxia has been shown to promote the development of CRC. In our previous study, it was shown that the mRNA and protein expression levels of TCF7L2 were elevated under hypoxia (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Several other investigations have confirmed that HIF-1α and HIF-2α are key regulators of responses to hypoxia in solid tumors (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Therefore, the current aimed to clarify the molecular mechanism by which hypoxia promoted TCF7L2 expression in CRC cells through RT-qPCR and western blotting in Caco-2 and HCT116 cells exposed to hypoxia. It was observed that the expression levels of HIF-1α were significantly enhanced, whereas those of HIF-2α were not altered, under hypoxia (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B), following 24 hours of hypoxic culture (1% O₂), HIF-1α mRNA and protein levels were significantly upregulated in Caco-2 and HCT-116 cells, time-course analysis confirmed maximal HIF-1α stabilization at 48 hours, aligning with the experimental duration used for subsequent assays. Consistently, the mRNA expression levels of HIF-1α and HIF-1α-specific downstream target genes [including vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT1)] in CRC cells were significantly upregulated under hypoxic conditions (24hours and 48 hours). Similarly, co-immunoprecipitation experiments confirmed that TCF7L2 interacted with HIF-1α in Caco-2 and HCT116 cells exposed to hypoxia (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). However, the physical interaction between HIF-1α and TCF7L2 could not be detected using the IP assay under normoxic conditions; this lack of interaction may be attributed to the low expression levels of HIF-1α in Caco-2 and HCT116 cells when exposed to normoxia (Fig. S1D). Based on the aforementioned results, the study assessed whether HIF-1α could directly bind to the TCF7L2 promoter, and regulate TCF7L2 gene transcription and expression. Using the online JASPAR platform, it was predicted that the HRE had a potential binding site on the TCF7L2 promoter region (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). In addition, a ChIP assay was carried out, which demonstrated significant fold enrichment of HIF-1α binding to the HRE of the TCF7L2 promoter under hypoxia in Caco-2 and HCT116 cells, accompanied with a considerable decrease in enrichment after successful HIF-1α knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Similarly, under normoxic conditions, the binding state between HIF-1α and TCF7L2 could not be detected using the ChIP assay (Fig. S1D). Finally, to determine whether HIF-1α could independently influence the observed increase expression of TCF7L2 following exposure to hypoxia, the Caco-2 and HCT-116 cells were transfected with HIF-1α shRNA to specifically knock down HIF-1α expression. The results indicated that HIF-1α knockdown significantly abolished the hypoxia-induced increase in TCF7L2 expression at the protein and mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF and G). Taken together, these results demonstrated that hypoxia may induce upregulation of TCF7L2 in a HIF-1α-dependent manner.\u003c/p\u003e \u003cp\u003e\u003cem\u003eUpregulation of TCF7L2 and HIF-1α are associated with poor clinicopathological features in patients with CRC.\u003c/em\u003e To explore the clinical significance of TCF7L2 in CRC, the mRNA levels of TCF7L2 were measured in 104 pairs of CRC specimens and adjacent normal colorectal tissues. The results revealed that the expression levels of TCF7L2 in CRC specimens were significantly higher than those in the matched adjacent normal colorectal specimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The present study also quantified the mRNA expression levels of HIF-1α in the 104 pairs of CRC specimens and adjacent normal colorectal tissues. Similarly, the mRNA expression levels of HIF-1α were upregulated in CRC tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Notably, a positive correlation was observed between HIF-1α expression and TCF7L2 expression in CRC specimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Consistently, elevated protein expression levels of HIF-1α and TCF7L2 were observed in 10 pairs of CRC tumor tissues than in adjacent normal colorectal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). To further elucidate the role of TCF7L2 in both clinical phenotypes and prognostic outcomes of CRC, immunohistochemical (IHC) staining was performed on tissue specimens from 104 CRC patients. Based on TCF7L2 protein expression levels, patients were stratified into high- and low-expression groups. Statistical analysis revealed a significant positive correlation between TCF7L2 overexpression and advanced T stage as well as distant metastasis (Table I). Survival curve analysis further demonstrated that high TCF7L2 expression was associated with poorer overall survival (OS) rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Subsequently, patients were categorized into two subgroups according to the combined expression patterns of HIF-1α and TCF7L2: the HIF-1α\u003csup\u003eHigh\u003c/sup\u003e/TCF7L2\u003csup\u003eHigh\u003c/sup\u003e group (n\u0026thinsp;=\u0026thinsp;44) and the HIF-1α\u003csup\u003eLow\u003c/sup\u003e/TCF7L2\u003csup\u003eLow\u003c/sup\u003e group (n\u0026thinsp;=\u0026thinsp;26). Clinicopathological correlation analysis indicated that the dual-high expression subgroup exhibited more aggressive features, including higher T stage and increased metastatic potential (Table II). Survival analysis further confirmed that patients in the dual-high group had significantly worse prognosis compared to those in the dual-low group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF), suggesting that co-detection of TCF7L2 and HIF-1α may serve as a prognostic biomarker panel for CRC. Representative IHC images depicting low and high expression patterns of both markers are shown in Supplementary Fig. S1E.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study elucidated the biological function and underlying mechanism of TCF7L2 in CRC. The initial findings demonstrated that TCF7L2 upregulation under hypoxic conditions is implicated in cell proliferation, migration, invasion and EMT progression in CRC \u003cem\u003ein vitro\u003c/em\u003e. In addition, TCF7L2 expression was associated with the maintenance of cancer stemness in CRC cells. Mechanistically, TCF7L2 was shown to promote CRC cell proliferation by activating the PI3K/AKT signaling pathway. Notably, TCF7L2 has been considered a key transcriptional regulator of HIF-1α, with HRE-binding sites located within the promoter region of HIF-1α, facilitating its transcriptional activation. The present \u003cem\u003ein vivo\u003c/em\u003e studies revealed that TCF7L2 enhanced tumor growth in nude mice. Furthermore, TCF7L2 mRNA and protein expression levels were elevated in CRC tissues compared with those in adjacent normal tissues, and this upregulation was associated with aberrant clinical features. Moreover, patients with CRC exhibiting elevated expression levels of TCF7L2 and HIF-1α had a poorer prognosis than those with lower expression levels of these markers.\u003c/p\u003e \u003cp\u003eDespite the development of ~\u0026thinsp;22 novel HIF-1α inhibitors and the completion of numerous preclinical studies over the past decade (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), these efforts have yet to yield clinically viable therapies. Several challenges may impede clinical translation, including insufficient inhibitory potency, limited specificity, suboptimal pharmacokinetic profiles, toxicity concerns and flaws in clinical trial design. In future research, we aim to investigate the potential synergistic effects of co-administering TCF7L2 inhibitors with HIF-1α inhibitors to enhance their tumor-suppressive properties. The present study may offer valuable insights for the clinical translation of HIF-1 inhibitors. Numerous studies have reported that TCF7L2 is elevated in carcinoma tissues and is associated with poor prognosis (\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Previous studies have also demonstrated that the TCF7L2 protein is primarily localized in the cell nuclei of gastric cancer (GC) tissues and the cytoplasm of adjacent tissues (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). These findings suggested that TCF7L2 may exert a cancer-promoting role in the nucleus of GC cells and high TCF7L2 expression could be significantly associated with a poor prognosis in patients with GC. Functionally, TCF7L2 acts as a key transcriptional regulator of the urokinase-type plasminogen activator receptor (uPAR, encoded by the PLAUR gene), directly binding to specific regulatory elements within the PLAUR promoter region to modulate its transcription, which suggests that TCF7L2 may serve a vital role in regulating gastric cancer MKN45 cell proliferation, anoikis resistance and migration (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Xiang \u003cem\u003eet al\u003c/em\u003e (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e) demonstrated that TCF7L2 can positively regulate aerobic glycolysis by suppressing EGLN2, leading to the upregulation of HIF-1α. This previous study also noted that TCF7L2 positively regulates HIF-1α stability and relevant glycolysis genes, such as GLUT1, hexokinase 2 and lactate dehydrogenase A in pancreatic cancer (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHypoxia is one of the most common and critical microenvironments in solid tumors. Various cellular responses to the hypoxic environment are regulated by a set of DNA-binding proteins, namely HIFs. As the predominant well-defined responsive regulator of hypoxic conditions in solid tumors, HIF-1α regulates multiple target genes through various biological pathways (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Previous research has reported that HIF-1α functions as a negative regulator of human arrest defective 1 (hARD1)-mediated β-catenin acetylation, and β-catenin is deacetylated under hypoxic conditions due to its competition with HIF-1α for hARD1 binding. hARD1 is involved in the HIF-1α-mediated, hypoxic inactivation of TCF4 (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). The present ChIP analysis revealed that TCF7L2 is an important downstream targeting gene for HIF-1α. Inconsistent with the present findings, Kaidi \u003cem\u003eet al\u003c/em\u003e (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e) reported that HIF-1α interacts with β-catenin via its amine group (NH2) terminal domain, interfering with the β-catenin-TCF7L2 association, suggesting a complex regulatory network between HIF-1α and TCF7L2.\u003c/p\u003e \u003cp\u003eEMT is a reversible process that was initially studied during embryo morphogenesis. In recent years, it has been shown that the state-switching between EMT and mesenchymal-epithelial transition serves a central role in various pathological processes, including tissue fibrosis, wound healing and the early stages of cancer development (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Numerous studies have demonstrated that EMT is an early event of tumor metastasis (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). During EMT, cancer cells undergo phenotypic changes and epithelial cells morphologically transform into mesenchymal cells, resulting in enhanced cell motility and invasion. Epithelial cells have a typical apical-basal polarity structure, and the tight, adherent and gap junctions between these cells limit their ability to migrate and invade. During EMT activation, epithelial cells lose cell polarity and cell-cell junctions, and gain the ability to invade and migrate, transforming into cells with a mesenchymal morphology and characteristics (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Given that the EMT and hypoxic microenvironment in tumors may share multiple signaling pathways, it has been suggested that hypoxia may induce EMT-like phenotypes in epithelial tumor cells (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Among all of the signaling pathways involved in tumor hypoxia stimulation, the HIF-1α pathway is one of the most important pathways for hypoxia-induced EMT. Li \u003cem\u003eet al\u003c/em\u003e (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e) reported that hypoxia enhances migration, activating EMT and promoting MMP expression in hepatocellular cancer cells by targeting the AKT and HIF-1α/VEGF signaling pathways. Maugeri \u003cem\u003eet al\u003c/em\u003e (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e) reported that overexpression of pituitary adenylate cyclase-activating polypeptide is associated with hypoxia-induced EMT activation by regulating an important EMT transcription factor, Zinc finger E-box-binding homeobox-1, in glioblastoma (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Shi \u003cem\u003eet al\u003c/em\u003e (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e) also demonstrated that PI3K/AKT signaling pathways are involved in hypoxia-induced EMT activation in CRC. The current study revealed that the mRNA and protein expression levels of HIF-1α and TCF7L2 were upregulated under the hypoxic tumor microenvironment in Caco-2 and HCT116 cell lines, which is in line with previous reports (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Furthermore, the migration and invasion of CRC cells were markedly enhanced after hypoxia stimulation. Notably, the epithelial marker E-cadherin was downregulated, whereas the mesenchymal markers vimentin and N-cadherin, and the EMT transcription factors Snail and Slug were significantly increased under hypoxia. However, TCF7L2 knockdown abrogated hypoxia-induced EMT activation in CRC. Taken together, these findings indicated that TCF7L2 may be involved in the hypoxia-induced EMT progression of CRC.\u003c/p\u003e \u003cp\u003eAccumulating evidence has shown that hypoxia has a notable role in the self-renewal and maintenance of stemness traits in CSCs and various carcinomas (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). CSCs have a high degree of metabolic adaptability and can survive in an oxygen-deficient environment. Additionally, the high acquisition and utilization of nutrients, such as glucose, enables CSCs to survive in restricted glucose level microenvironments, thereby promoting cell survival and tumorigenic potential (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). CSCs possess intrinsic stem cell characteristics, including self-renewal and resistance to chemotherapeutic agents. The capacity for tumor self-renewal is recognized as a defining hallmark of CSCs, contributing to tumor recurrence, drug resistance and metastasis. The sphere formation assay is a widely accepted technique for evaluating the formation of CSC-like cells. In this assay, single cells isolated from tumor spheres have demonstrated the ability to generate tumor spheres, thereby indicating the self-renewal capacity of cancer stem-like cells. The present study demonstrated that HCT-16 cells exhibited sphere-forming capability, which was sustained through the second passage under hypoxia, suggesting the important role of TCF7L2 in CRC stemness maintenance. Moreover, the results revealed that some typical stemness-associated genes, such as CD44, CD133, ALDH1A1, EpCAM, Nanog and OCT4, were significantly enriched in CRC cells after hypoxic stimulation. Conversely, TCF7L2 knockdown exhibited the opposite effects. Both CD44 and CD133 (also known as prominin-1) are putative stem markers for isolating CSCs from CRC (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). Therefore, the CD44\u003csup\u003e+\u003c/sup\u003e/CD133\u003csup\u003e+\u003c/sup\u003e subpopulation (defined as CRC CSCs) was isolated to further explore whether TCF7L2 was involved in hypoxia facilitating the development of CRC through enhanced stemness of CRC CSCs. The results demonstrated the independent role of TCF7L2 in cancer stemness maintenance of CRC.\u003c/p\u003e \u003cp\u003ePrevious research has demonstrated that β-catenin, a pivotal component of the Wnt signaling pathway, engages with HIF-1α across a range of physiological and pathological contexts, with the activation of the Wnt signaling pathway contributing to chemotherapy resistance (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). The present study identified an interaction between TCF7L2 and HIF-1α in CRC cells under hypoxic conditions. Given these findings, it is pertinent to investigate whether TCF7L2 facilitates HIF-1α in modulating the malignant behavior of CRC. Additionally, the PI3K/Akt pathway is integral to the regulation of metabolic processes, encompassing both matter and energy metabolism. The upregulation of HIF-1α expression and the enhancement of glycolysis in tumor cells are contingent upon the activation of the PI3K/Akt signaling pathway. Nevertheless, the precise mechanisms underlying this relationship remain inadequately understood. In light of these observations, our research aimed to investigate whether the PI3K/Akt pathway serves as a crucial mechanism through which TCF7L2 modulates the malignant behavior of CRC under hypoxic conditions. To date, the precise regulatory mechanism of TCF7L2 in CRC remains unclear. The present study identified a significant association between TCF7L2 expression and the activation of the PI3K/AKT signaling pathway. The PI3K/AKT signaling pathway is known to serve a crucial role in various biological processes, including cell proliferation, apoptosis and cell cycle progression. This pathway has also been reported to mediate the maintenance of stemness in various types of cancer, including liver cancer and CRC (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). The current study indicated that TCF7L2 may exert a proliferative effect on CRC cells by activating the PI3K/AKT signaling pathway.\u003c/p\u003e \u003cp\u003eIn conclusion, the findings of the present study demonstrated that TCF7L2 may serve a critical role in the progression of CRC. TCF7L2 upregulation was positively associated with poor clinical features in patients with CRC. Furthermore, to the best of our knowledge, the current study demonstrated previously unreported mechanistic crosstalk between HIF-1α and TCF7L2, indicating that TCF7L2 functions as a direct downstream target of HIF-1α in mediating tumor survival, metastasis and the maintenance of stemness properties in CRC. This study provides a theoretical basis for considering TCF7L2 and HIF-1α as potential therapeutic targets for CRC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo\u0026nbsp;funding\u0026nbsp;was received.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated in the present study may be requested from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKT, YC, JPG, and YL were instrumental in developing the study concepts and design. The literature review was conducted by YC, JPG, and YL. Experimental procedures were carried out by KT. Data analysis was performed by YC, JPG, and YL. The manuscript was written and revised collaboratively by KT, YC, JPG, and YL. The authenticity of all raw data has been verified by YC, JPG, and YL.\u0026nbsp;All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study was approved by the Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University (Chongqing, China, ethical approval no. 2023-326). Written informed consent was obtained from all participants or their parents.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from patients or their families for publication of pathological sections or tissue sample images included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSiegel RL, Giaquinto AN, Jemal A: Cancer statistics, 2024. CA Cancer J Clin. 74:12\u0026ndash;49, 2024.\u003c/li\u003e\n \u003cli\u003eAndrei P, Battuello P, Grasso G, Rovera E, Tesio N, Bardelli A: Integrated approaches for precision oncology in colorectal cancer: The more you know, the better. Semin Cancer Biol 84: 199\u0026ndash;213, 2022.\u003c/li\u003e\n \u003cli\u003eValentini V, Coco C, Gambacorta MA, Barba MC, Meldolesi E: Evidence and research perspectives for surgeons in the European Rectal Cancer Consensus Conference (EURECA-CC2). Acta Chir Iugosl 57: 9\u0026ndash;16, 2010.\u003c/li\u003e\n \u003cli\u003eSiegel R, DeSantis C, Virgo K, Stein K, Mariotto A, Smith T, et al: Cancer treatment and survivorship statistics, 2012. CA Cancer J Clin 62: 220\u0026ndash;241, 2012.\u003c/li\u003e\n \u003cli\u003eChen Z, Han F, Du Y, Shi H, Zhou W. Hypoxic microenvironment in cancer: molecular mechanisms and therapeutic interventions. Signal Transduct Target Ther 8: 70, 2023.\u003c/li\u003e\n \u003cli\u003eWicks, EE, Semenza, GL. Hypoxia-inducible factors: cancer progression and clinical translation. J Clin Invest 132: e159839, 2022.\u003c/li\u003e\n \u003cli\u003eSch\u0026ouml;del J, Grampp S, Maher ER, et al. Hypoxia, Hypoxia-inducible Transcription Factors, and Renal Cancer. Eur Urol. 69:646-657, 2016.\u003c/li\u003e\n \u003cli\u003eCowman SJ, Koh MY. Revisiting the HIF switch in the tumor and its immune microenvironment. Trends Cancer 8: 28-42, 2022.\u003c/li\u003e\n \u003cli\u003eSchito L, Semenza GL: Hypoxia-Inducible Factors: Master Regulators of Cancer Progression. Trends cancer 2: 758\u0026ndash;770, 2016.\u003c/li\u003e\n \u003cli\u003eSemenza GL: Targeting HIF-1 for cancer therapy. Nat Rev Cancer 3: 721\u0026ndash;732, 2003.\u003c/li\u003e\n \u003cli\u003ede Heer EC, Jalving M, Harris AL. HIFs, angiogenesis, and metabolism: elusive enemies in breast cancer. J Clin Invest 130: 5074\u0026ndash;5087, 2020.\u003c/li\u003e\n \u003cli\u003eWilson GK, Tennant DA, McKeating JA: Hypoxia inducible factors in liver disease and hepatocellular carcinoma: current understanding and future directions. J Hepatol 61:1397\u0026ndash;1406, 2014.\u003c/li\u003e\n \u003cli\u003eLu T, Tang J, Shrestha B, Heath BR, Hong L, Lei YL, et al: Up-regulation of hypoxia-inducible factor antisense as a novel approach to treat ovarian cancer. Theranostics 10:6959\u0026ndash;6976, 2020.\u003c/li\u003e\n \u003cli\u003eZeng L, Zhou HY, Tang NN, Zhang WF, He GJ, Hao B, et al: Wortmannin influences hypoxia-inducible factor-1 alpha expression and glycolysis in esophageal carcinoma cells. World J Gastroenterol 22: 4868\u0026ndash;4880, 2016.\u003c/li\u003e\n \u003cli\u003eNagaraju GP, Bramhachari PV, Raghu G, El-Rayes BF: Hypoxia inducible factor-1\u0026alpha;: Its role in colorectal carcinogenesis and metastasis. Cancer Lett 366: 11\u0026ndash;18, 2015.\u003c/li\u003e\n \u003cli\u003eDel Bosque-Plata L, Mart\u0026iacute;nez-Mart\u0026iacute;nez E, Espinoza-Camacho M\u0026Aacute;, Gragnoli C: The Role of TCF7L2 in Type 2 Diabetes. Diabetes 70: 1220\u0026ndash;1228, 2021.\u003c/li\u003e\n \u003cli\u003eZhang T, Wang B, Su F, Gu B, Xiang L, Gao L, et al: TCF7L2 promotes anoikis resistance and metastasis of gastric cancer by transcriptionally activating PLAUR. Int J Biol Sci 18: 4560\u0026ndash;4577, 2022.\u003c/li\u003e\n \u003cli\u003eIshiguro H, Wakasugi T, Terashita Y, Sakamoto N, Tanaka T, Sagawa H, et al: Nuclear expression of TCF4/TCF7L2 is correlated with poor prognosis in patients with esophageal squamous cell carcinoma. Cell Mol Biol Lett 21: 5, 2016.\u003c/li\u003e\n \u003cli\u003eRice SJ, Liu X, Hyland V, Liu Z, Belani CP: Mutations in genes connected with the TCF7L2 transcription factor are associated with a poor prognosis in non-small cell lung cancer. Lung Cancer 141: 97\u0026ndash;100, 2020.\u003c/li\u003e\n \u003cli\u003eGrant SFA. The TCF7L2 Locus: A Genetic Window Into the Pathogenesis of Type 1 and Type 2 Diabetes. Diabetes Care 42: 1624-1629, 2019.\u003c/li\u003e\n \u003cli\u003eChen CS, Huang CY, Huang SP, et al. Genetic interaction analysis of TCF7L2 for biochemical recurrence after radical prostatectomy in localized prostate cancer. Int J Med Sci 12: 243-247, 2015.\u003c/li\u003e\n \u003cli\u003eFolsom AR, Pankow JS, Peacock JM, Bielinski SJ, Heiss G, Boerwinkle E: Variation in TCF7L2 and increased risk of colon cancer: the Atherosclerosis Risk in Communities (ARIC) Study. Diabetes Care 31: 905\u0026ndash;909, 2008.\u003c/li\u003e\n \u003cli\u003eGiles RH, Lolkema MP, Snijckers CM, Belderbos M, van der Groep P, Mans DA, et al: Interplay between VHL/HIF1alpha and Wnt/beta-catenin pathways during colorectal tumorigenesis. Oncogene 25: 3065\u0026ndash;3070, 2006.\u003c/li\u003e\n \u003cli\u003eShi J, Lv Q, Miao D, Xiong Z, Wei Z, Wu S, et al: HIF2\u0026alpha; Promotes Cancer Metastasis through TCF7L2-Dependent Fatty Acid Synthesis in ccRCC. Research (Washington, D.C.) 7: 0322, 2024.\u003c/li\u003e\n \u003cli\u003eChang J, Tian J, Yang Y, et al. A Rare Missense Variant in TCF7L2 Associates with Colorectal Cancer Risk by Interacting with a GWAS-Identified Regulatory Variant in the MYC Enhancer. Cancer Res 78: 5164-5172, 2018.\u003c/li\u003e\n \u003cli\u003eTang K, Toyozumi T, Murakami K, Sakata H, Kano M, Endo S, et al: HIF-1\u0026alpha; stimulates the progression of oesophageal squamous cell carcinoma by activating the Wnt/\u0026beta;-catenin signalling pathway. Br J Cancer 127: 474\u0026ndash;487, 2022.\u003c/li\u003e\n \u003cli\u003eLivak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-408,2001.\u003c/li\u003e\n \u003cli\u003eFarina AR, Cappabianca L, Sebastiano M, Zelli V, Guadagni S, Mackay AR. Hypoxia-induced alternative splicing: the 11th Hallmark of Cancer. J Exp Clin Cancer Res. 39:110, 2020.\u003c/li\u003e\n \u003cli\u003eKessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell.141:52-67, 2010.\u003c/li\u003e\n \u003cli\u003eChen X, Zeh HJ, Kang R, Kroemer G, Tang D. Cell death in pancreatic cancer: from pathogenesis to therapy. Nat Rev Gastroenterol Hepatol. 18:804-823,2021.\u003c/li\u003e\n \u003cli\u003eDuan S, Huang W, Liu X, et al. IMPDH2 promotes colorectal cancer progression through activation of the PI3K/AKT/mTOR and PI3K/AKT/FOXO1 signaling pathways. J Exp Clin Cancer Res. 37:304, 2018.\u003c/li\u003e\n \u003cli\u003eDong S, Liang S, Cheng Z, et al. ROS/PI3K/Akt and Wnt/\u0026beta;-catenin signalings activate HIF-1\u0026alpha;-induced metabolic reprogramming to impart 5-fluorouracil resistance in colorectal cancer. J Exp Clin Cancer Res. 41:15, 2022.\u003c/li\u003e\n \u003cli\u003eChen Y, Xu X, Wang Y, et al. Hypoxia-induced SKA3 promoted cholangiocarcinoma progression and chemoresistance by enhancing fatty acid synthesis via the regulation of PAR-dependent HIF-1a deubiquitylation. J Exp Clin Cancer Res. 42:265, 2023.\u003c/li\u003e\n \u003cli\u003eZhang X, Yang L, Lei W, Hou Q, Huang M, Zhou R, et al: Single-cell sequencing reveals CD133+CD44--originating evolution and novel stemness related variants in human colorectal cancer. EBioMedicine 82: 104125, 2022.\u003c/li\u003e\n \u003cli\u003eKoukourakis MI, Giatromanolaki A, Skarlatos J, et al. Hypoxia inducible factor (HIF-1a and HIF-2a) expression in early esophageal cancer and response to photodynamic therapy and radiotherapy. Cancer Res. 61:1830-1832, 2001.\u003c/li\u003e\n \u003cli\u003eBoddy JL, Fox SB, Han C, et al. The androgen receptor is significantly associated with vascular endothelial growth factor and hypoxia sensing via hypoxia-inducible factors HIF-1a, HIF-2a, and the prolyl hydroxylases in human prostate cancer. Clin Cancer Res.11:7658-7663, 2005.\u003c/li\u003e\n \u003cli\u003eLiu J, Gao Y, Zhang X. A patent review on hypoxia-inducible factor (HIF) modulators (2021-2023). Expert Opin Ther Pat.34:651-664, 2024.\u003c/li\u003e\n \u003cli\u003eDel Bosque-Plata L, Hern\u0026aacute;ndez-Cort\u0026eacute;s EP, Gragnoli C. The broad pathogenetic role of TCF7L2 in human diseases beyond type 2 diabetes. J Cell Physiol 237: 301\u0026ndash;312, 2022.\u003c/li\u003e\n \u003cli\u003eWu Q, Ma J, Wei J, Meng W, Wang Y, Shi M: lncRNA SNHG11 Promotes Gastric Cancer Progression by Activating the Wnt/\u0026beta;-Catenin Pathway and Oncogenic Autophagy. Mol Ther 29: 1258\u0026ndash;1278, 2021.\u003c/li\u003e\n \u003cli\u003eWang D, Wu S, He J, Sun L, Zhu H, Zhang Y, et al: FAT4 overexpression promotes antitumor immunity by regulating the \u0026beta;-catenin/STT3/PD-L1 axis in cervical cancer. J Exp Clin Cancer Res 42: 222, 2023.\u003c/li\u003e\n \u003cli\u003eZhang T, Wang B, Su F, et al. TCF7L2 promotes anoikis resistance and metastasis of gastric cancer by transcriptionally activating PLAUR. Int J Biol Sci. 18:4560-4577, 2022.\u003c/li\u003e\n \u003cli\u003eGe L, Zhao G, Lan C, et al. MESP2 binds competitively to TCF4 to suppress gastric cancer progression by regulating the SKP2/p27 axis. Cell Death Discov. 9:79, 2023.\u003c/li\u003e\n \u003cli\u003eXiang J, Hu Q, Qin Y, Ji S, Xu W, Liu W, et al: TCF7L2 positively regulates aerobic glycolysis via the EGLN2/HIF-1\u0026alpha; axis and indicates prognosis in pancreatic cancer. Cell Death Dis 9: 321, 2018.\u003c/li\u003e\n \u003cli\u003eYuan X, Ruan W, Bobrow B, Carmeliet P, Eltzschig HK: Targeting hypoxia-inducible factors: therapeutic opportunities and challenges. Nat Rev Drug Discov 23: 175\u0026ndash;200, 2024.\u003c/li\u003e\n \u003cli\u003eLim JH, Chun YS, Park JW: Hypoxia-inducible factor-1alpha obstructs a Wnt signaling pathway by inhibiting the hARD1-mediated activation of beta-catenin. Cancer Res 68: 5177\u0026ndash;5184, 2008.\u003c/li\u003e\n \u003cli\u003eKaidi A, Williams AC, Paraskeva C: Interaction between beta-catenin and HIF-1 promotes cellular adaptation to hypoxia. Nat Cell Biol 9: 210\u0026ndash;217, 2007.\u003c/li\u003e\n \u003cli\u003ePastushenko I, Blanpain C: EMT Transition States during Tumor Progression and Metastasis. Trends Cell Biol 29: 212\u0026ndash;226, 2019.\u003c/li\u003e\n \u003cli\u003eAkhmetkaliyev A, Alibrahim N, Shafiee D, Tulchinsky E. EMT/MET plasticity in cancer and Go-or-Grow decisions in quiescence: the two sides of the same coin?. Mol Cancer. 22:90, 2023.\u003c/li\u003e\n \u003cli\u003eHuang Y, Hong W, Wei X. The molecular mechanisms and therapeutic strategies of EMT in tumor progression and metastasis. J Hematol Oncol. 15:129, 2022.\u003c/li\u003e\n \u003cli\u003eDavis FM, Stewart TA, Thompson EW, Monteith GR: Targeting EMT in cancer: opportunities for pharmacological intervention. Trends Pharmacol Sci 35: 479\u0026ndash;488, 2014.\u003c/li\u003e\n \u003cli\u003eHapke RY, Haake SM: Hypoxia-induced epithelial to mesenchymal transition in cancer. Cancer Lett 487: 10\u0026ndash;20, 2020.\u003c/li\u003e\n \u003cli\u003eLi C, Wang Q, Shen S, Wei X, Li G: HIF-1\u0026alpha;/VEGF signaling-mediated epithelial-mesenchymal transition and angiogenesis is critically involved in anti-metastasis effect of luteolin in melanoma cells. Phytother Res 33: 798\u0026ndash;807, 2019.\u003c/li\u003e\n \u003cli\u003eMaugeri G, D\u0026apos;Amico AG, Saccone S, Federico C, Ras\u0026agrave; DM, Caltabiano R, et al: Effect of PACAP on Hypoxia-Induced Angiogenesis and Epithelial-Mesenchymal Transition in Glioblastoma. Biomedicines 9: 965, 2021.\u003c/li\u003e\n \u003cli\u003eShi Z, To SKY, Zhang S, Deng S, Artemenko M, Zhang M, et al. Hypoxia-induced Nur77 activates PI3K/Akt signaling via suppression of Dicer/let-7i-5p to induce epithelial-to-mesenchymal transition. Theranostics. 11:3376\u0026ndash;3391, 2021.\u003c/li\u003e\n \u003cli\u003eZhang Q, Bai X, Chen W, et al. Wnt/\u0026beta;-catenin signaling enhances hypoxia-induced epithelial-mesenchymal transition in hepatocellular carcinoma via crosstalk with hif-1\u0026alpha; signaling. Carcinogenesis. 34:962-973, 2013.\u003c/li\u003e\n \u003cli\u003eKeith B, Simon MC. Hypoxia-inducible factors, stem cells, and cancer. Cell. 129:465-472, 2007.\u003c/li\u003e\n \u003cli\u003eZhang D, Yang L, Liu X, et al. Hypoxia modulates stem cell properties and induces EMT through N-glycosylation of EpCAM in breast cancer cells. J Cell Physiol. 235:3626-3633, 2020.\u003c/li\u003e\n \u003cli\u003eSun X, Lv X, Yan Y, Zhao Y, Ma R, He M, et al: Hypoxia-mediated cancer stem cell resistance and targeted therapy. Biomed Pharmacother 130: 110623, 2020.\u003c/li\u003e\n \u003cli\u003eWei F, Zhang T, Deng SC, Wei JC, Yang P, Wang Q, et al: PD-L1 promotes colorectal cancer stem cell expansion by activating HMGA1-dependent signaling pathways. Cancer Lett 450: 1\u0026ndash;13, 2019.\u003c/li\u003e\n \u003cli\u003eBoso D, Rampazzo E, Zanon C, et al. HIF-1\u0026alpha;/Wnt signaling-dependent control of gene transcription regulates neuronal differentiation of glioblastoma stem cells. Theranostics. 2019;9(17):4860-4877.\u003c/li\u003e\n \u003cli\u003eStefani C, Miricescu D, Stanescu-Spinu II, et al. Growth Factors, PI3K/AKT/mTOR and MAPK Signaling Pathways in Colorectal Cancer Pathogenesis: Where Are We Now?. Int J Mol Sci. 22:10260, 2021.\u003c/li\u003e\n \u003cli\u003eTian LY, Smit DJ, J\u0026uuml;cker M. The Role of PI3K/AKT/mTOR Signaling in Hepatocellular Carcinoma Metabolism. Int J Mol Sci.24:2652, 2023.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable I. Association between TCF7L2 expression and the clinicopathological features of patients with colorectal cancer.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eTCF7L2 expression level\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.745\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge, years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.644\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026le;65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.027\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.781\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.011\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePathological stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.141\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003eP\u0026lt;0.05. TCF7L2, transcription factor 7-like 2.\u003c/p\u003e \u003cp\u003eTable II. Association among TCF7L2, expression HIF-1α expression and the clinicopathological features of patients with colorectal cancer.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003eHIF-1α/TCF7L2 expression\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHIF-1α\u003csup\u003eHigh\u003c/sup\u003e TCF7L2 \u003csup\u003eHigh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHIF-1α\u003csup\u003eHigh\u003c/sup\u003e TCF7L2 \u003csup\u003eLow\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHIF-1α\u003csup\u003eLow\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTCF7L2 \u003csup\u003eHigh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHIF-1α\u003csup\u003eLow\u003c/sup\u003e TCF7L2 \u003csup\u003eLow\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.906\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge, years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" 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HIF, hypoxia-inducible factor; TCF7L2, transcription factor 7-like 2.\u003c/p\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":"transcription factor 7-like 2, hypoxia, colorectal cancer, epithelial-mesenchymal transition, cancer stem cell","lastPublishedDoi":"10.21203/rs.3.rs-7002021/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7002021/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHypoxia plays a crucial role in the advancement of colorectal cancer (CRC); however, the downstream mechanisms facilitated by hypoxia-inducible factor 1α (HIF-1α) remain incompletely understood. This study employed \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models to investigate the role of transcription factor 7-like 2 (TCF7L2) under hypoxic conditions in CRC. Utilizing reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, we observed an upregulation of TCF7L2 mRNA and protein expression in Caco-2 and HCT-116 CRC cell lines under hypoxia. Functional assays, including CCK-8, colony and sphere formation, Transwell, flow cytometry, and xenograft tumor models, provided evidence that the knockdown of TCF7L2 leads to the suppression of CRC cell proliferation, the induction of apoptosis, cell cycle arrest at the G0/G1 phase, and a decrease in migration and invasion capabilities. Furthermore, it inhibited epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) characteristics \u003cem\u003ein vitro\u003c/em\u003e, while also reducing tumor growth \u003cem\u003ein vivo\u003c/em\u003e. Mechanistically, chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (co-IP) assays have elucidated that the expression of TCF7L2 induced by hypoxia is dependent on HIF-1α, which directly binds to hypoxia response elements (HREs) within the TCF7L2 promoter. Additionally, Western blot and experiments employing the PI3K inhibitor LY294002 have demonstrated that TCF7L2 activates the PI3K/AKT signaling pathway, thereby facilitating the proliferation of CRC cells. A clinical analysis of 104 CRC specimens, utilizing immunohistochemistry (IHC) and RT-qPCR, revealed that elevated expression levels of TCF7L2 were significantly associated with advanced T stage, metastasis, and unfavorable prognosis. Spearman correlation analysis confirmed a positive relationship between the expressions of TCF7L2 and HIF-1α, while Kaplan-Meier survival analysis demonstrated that their co-expression was predictive of reduced overall survival. Collectively, these findings position TCF7L2 as a critical downstream effector of HIF-1α in CRC, underscoring its potential as a therapeutic target for addressing hypoxic tumor microenvironments.\u003c/p\u003e","manuscriptTitle":"Mechanistic insights into hypoxia-induced TCF7L2 upregulation and its oncogenic effects on colorectal cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-03 15:08:58","doi":"10.21203/rs.3.rs-7002021/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":"49f16e99-e5ff-47d4-b8c8-3b7c780d315c","owner":[],"postedDate":"July 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-14T09:25:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-03 15:08:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7002021","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7002021","identity":"rs-7002021","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}