TCF7L2 promotes tumor progression by regulating hypoxia-inducible factor 1 alpha through activating the PI3K/AKT signaling pathway in colorectal carcinoma

TCF7L2 promotes tumor progression by regulating hypoxia-inducible factor 1 alpha through activating the PI3K/AKT signaling pathway in colorectal carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article TCF7L2 promotes tumor progression by regulating hypoxia-inducible factor 1 alpha through activating the PI3K/AKT signaling pathway in colorectal carcinoma Yong Cheng, Kang Tang, Jianping Gong, Yang Li This is a preprint; it has not been peer reviewed by a journal.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-4860804/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 is a critical pathogenic factor in cancer development and metastasis. The pivotal role of hypoxia-inducible factor 1α (HIF-1α) in tumor progression under hypoxic conditions is well-documented. However, the specific mechanisms by which HIF-1α contributes to colorectal cancer (CRC) progression remain inadequately elucidated. In this study, we observed an upregulation of Transcription Factor 7-like 2 (TCF7L2) in CRC cells under hypoxic conditions. Meanwhile, hypoxia-induced overexpression of TCF7L2 plays a pivotal role in the proliferation, apoptosis, cell cycle arrest, migration, invasion, epithelial-mesenchymal transition (EMT), and cancer stem cell (CSC) characteristics of colorectal cancer (CRC) cells in vitro. Additionally, our findings indicate that the inhibition of TCF7L2 results in a significant reduction of tumor growth in vivo. Mechanistically, hypoxia-induced up-regulation of TCF7L2 expression occurs in a HIF-1α-dependent manner. Chromatin immunoprecipitation (ChIP) assays demonstrated increased HIF-1α binding to the promoter sequence of TCF7L2 following hypoxic stimulation. Furthermore, our findings indicate that TCF7L2 plays an oncogenic role in colorectal cancer (CRC) by activating the PI3K/AKT signaling pathway. Additionally, we observed that elevated expression levels of both HIF-1α and TCF7L2 in CRC specimens are associated with aberrant clinicopathological features. Co-expression of TCF7L2 and HIF-1α predicts a poor prognosis in CRC patients. Targeting TCF7L2 is a promising approach to colorectal cancer therapy. Biological sciences/Cancer/Gastrointestinal cancer/Colorectal cancer Health sciences/Biomarkers/Prognostic markers 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 among the most prevalent solid malignancies and represents the third leading cause of tumor-related mortality worldwide, with the third highest disease-specific death rate [ 1 ]. Although multiple treatments such as whole genome sequencing (WGS) analysis, total mesorectal excision (TME), neoadjuvant chemotherapy or chemoradiotherapy and multidisciplinary team management (MDT) have been rapidly developed and widely applied in clinics for past decades [ 2 ]. Unfortunately, combination treatment including targeted therapy for advanced CRC has limited effectiveness, patients with advanced colorectal cancer still have poor survival, with 5-year survival rate is only about 12% [ 3 , 4 ]. Therefore, in recent years, mounting studies had focused on identifying the biological molecular mechanism underlying CRC initiation and metastasis. These urgently prompted us to explore novel effective molecular therapeutics targets to improve survival in patients with colorectal cancer. Hypoxia in one of the most common and critical microenvironments during solid tumors progression [ 5 ]. It has been reported that hypoxia-inducible factors participate in proliferation, inflammation reaction, angiogenesis, invasion and distant metastasis in various types of solid carcinoma [ 6 ]. In response to the hypoxic cellular microenvironment, cancer cells undergo a complex series of adaptive changes including cell energy metabolism, proliferation, apoptosis, invasion and angiogenesis. Hypoxia inducible factor-1 (HIF-1) are predominant responsive regulator to hypoxia during tumor progression, which is a heterodimer consisting of α and β subunits. Many studies showed that in the presence of hypoxic microenvironment, HIF-1α evades the degradation by inhibition of the hydroxylation of PHD and combined with HIF-1β subunit to form heterodimer, translocate from the cytoplasm into the nucleus, they interact with specific DNA sequences contain hypoxia response elements (HRE), and mediates the transcription and expression of multiple downstream target genes [ 7 ]. To date, over 100 HIF downstream target genes have been discovered, most of these genes involved in the metastasis cascade, including epithelial–mesenchymal transition (EMT), extracellular matrix (ECM), enhanced tumor cell motility, angiogenesis [ 8 ]. Clinically, in most solid carcinoma types including breast cancer [ 9 ], hepatocellular carcinoma [ 10 ], ovarian cancer [ 11 ], esophageal cancer [ 12 ] and colorectal cancer [ 13 ], elevated expression of HIF-1α was identified to positively associated with poor prognosis. TCF7L2, also known as Transcription factor 7-like 2, which has been shown directly regulates genes involved in metabolism and cell cycle control within adipocytes by genome-wide analysis of TCF7L2 binding and gene expression [ 14 ]. Recently, TCF7L2 had been discovered expressing in various types of non-mineralizing soft cancerous tissues including colon, esophageal, lung, skin and stomach, suggesting TCF7L2 might play an essential role in the carcinogenesis of malignant tumors[ 15 – 17 ]. It was reported that TCF7L2 gene was associated with type 2 diabetes, but it is not associated with cancer, except in reverse with prostate cancer, in nondiabetic participants, the association between TCF7L2 and colon cancer was still observed [ 18 ]. Moreover, a previously study indicated that TCF7L2 was found overexpression in mammary epithelial cell-derived organoids and involved in EMT, more importantly, cellular abnormal expression of TCF7L2 protein was positively associated with increased expression level of HIF-1α [ 19 ]. Similarity, in clear cell renal cell carcinoma (ccRCC), hypoxia microenvironment was reported to increase the expression of TCF7L2 in a HIF-2α dependent manner, and Hypoxia regulated TCF7L2 high expression also participated in ccRCC tumor survival and distant metastasis [ 20 ]. However, to the best of our knowledge, there are few investigations focus on the function of TCF7L2 in CRC. Our study initially identified an elevated expression level of TCF7L2 in colorectal cancer (CRC) both in vivo and in vitro. Furthermore, overexpression of TCF7L2 in CRC patients was found to correlate with advanced clinical stages and metastasis. In addition, this research represents the first investigation into the relationship between TCF7L2 expression and CRC cell proliferation, survival, epithelial-mesenchymal transition (EMT), and the maintenance of cancer stemness. Finally, we explored the molecular mechanisms underlying the involvement of TCF7L2 in CRC progression. Materials and methods Clinical specimens and cell culture 104 Clinical CRC samples and adjacent non-tumor samples were gained from the Second Affiliated Hospital of Chongqing Medical University (Chongqing, China) from 2009–2013. No patients received preoperative radiotherapy or chemotherapy before surgery. All patients in this study provided informed written consent and approved by the Ethical Committee of the Second Affiliated Hospital of Chongqing Medical University. CRC cell lines Caco-2, HCT-116, HT-29, LoVo, SW480, SW620 and the human immortalized normal small intestine epithelium cell lines HIEC-6 were obtained from the American Type Culture Collection (ATCC). All CRC cell lines were cultured in Leibovitz’s L-15 medium (Gibco, CA, USA) with 10% FBS (Thermo Fisher Scientific) at 37˚C and 5% CO 2 . HIEC-6 cell lines were cultured in Opti-MEM I Reduced Serum Medium (Gibco) supplied with 4% FBS (Thermo Fisher Scientific) and 10 ng/mL Epidermal Growth Factor (Sigma, St Louis, USA) at 37 ˚C and 5% CO 2 . To mimic the hypoxic microenvironment, cells were cultured in hypoxic cell incubator (Thermo Fisher Scientific) supplied with 1% O 2 , 5% CO 2 and 94% N 2 . RNA interference The TCF7L2 shRNA (h) Lentiviral Particles (Santa Cruz, SC-43525-V) or HIF-1α shRNA (h) Lentiviral Particles (Santa Cruz, sc-35561-V) were transfected in Caco-2 and HCT-116 cells to establish stable knock down of TCF7L2 or HIF-1α. Generally, CRC cell lines (5×10 4 /well) were plated in a 6-well plate 24h prior to lentiviral particles transfection. Then the thawed lentiviral particles were transfected into cells with a multiplicity of infection (MOI = 4) co-cultured with 5 µg/ml polybrene (Santa Cruz). Stable clones expressing the TCF7L2 or HIF-1α shRNA were selected by using 10 µg/ml Puromycin dihydrochloride (Sigma) for 14 days. Control shRNA Lentiviral Particles (Santa Cruz, sc-108080) were transduced into cells as a negative control. The TCF7L2 Lentiviral Activation Particles (h) (Santa Cruz, sc-400607-LAC) was transfected in Caco-2 and HCT-116 cells to establish stable over-expression of TCF7L2 cell lines. Control Lentiviral Activation Particles (Santa Cruz, sc-437282) were transduced into cells as a negative control. The specific operation procedure and method was referenced in previous studies [ 21 ]. RNA extraction and quantitative real-time PCR (qRT-PCR) Total RNA from tissues or cultured CRC cells was isolated with RNAiso plus (TaKaRa) according to the manufacture’s procedures. cDNA was synthesized with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The qRT-PCR analyses were conducted according to a previously described. The primer sequences designed for qRT-PCR analysis were listed in Supplementary Table S1 . All reactions were performed in triplicate. The specific experimental methods were previously published [ 21 ]. Western-blot analysis Proteins were extracted from frozen tissues or cultured CRC cells with phosphatase inhibitor contained RIPA solution (Sigma). 20 µg protein samples were subjected by SDS-PAGE gel and transferred onto a PVDF membrane. After blocked in 3% Bovine Serum Albumins (BSA) diluted with 0.1% TBST solution at room temperature for 1h. Then membranes were incubated at 4°C overnight with the primary Antibodies, the information of all primary antibodies used in this study is provided in Supplementary Table S2 . Subsequently, the membranes were incubated at room temperature for 1h with anti-rabbit IgG HRP secondary Ab (1:2000 dilution, sc‐2301; Santa Cruz) or anti‐mouse IgG HRP secondary Ab (1:2000 dilution, sc‐2005; Santa Cruz). The specific experimental steps refer to the previous study [ 21 ]. Cell proliferation, colony formation and sphere formation assay The cell proliferation assay was carried out using the Cell Counting Kit-8 (CCK-8; Dojindo, Japan) at indicated time points according to the manufactures protocol. Colony-formation assays were performed as follows. Briefly, 1,000 cells/well were plated into a sex-well plate and cultured in complete cell medium for 12 days. The colonies in each group were then fixed and stained with Diff-Quick kit (Sysmex, Kobe, Japan). The colony number was only counted by containing at least of 50 cells/colony. Plating efficiency (%) = the number of colonies formed / the number of cells seeded ×100. For sphere formation assay, 1 × 10 4 cells were cultured on low-adherence plates in 10 mL serum-free medium with supplements of epidermal growth factor (EGF, 10 ng/ml), basic fibroblast growth factor (bFGF, 20 ng/ml), 4% B27 and 4 µg/ml insulin. Sphere formation efficiency (SFE, %) = the number of spheres formed at 14th day / the number of cells seeded ×100. The specific experimental methods were previously published [ 21 ]. Trans well migration and invasion assay In vitro migration (without Matrigel pre-coated) assays and invasion (with Matrigel pre-coated) were performed using 24-well trans-well plates with 6.5 mm diameter and 8 µm pores filters (Corning). Briefly, CRC cells (2 × 10 5 ) were plated into the upper chamber, trans-well plates were then incubated for 24h (migration) or 30h (invasion) at 37°C, respectively. Finally, migrated or invaded cells attached on the lower chamber were then gently washed with PBS, fixed and stained with Diff-Quick stain kit (Sysmex, Kobe, Japan). The procedure of the experiment was conducted by referring to previous studies [ 21 ]. Cell apoptosis and cell cycle analysis Cell apoptosis analysis was performed by using the annexin V–FITC Apoptosis Detection Kit (BD Biosciences) following the manufacturer’s protocol. Briefly, CRC cells (5 × 10 5 cells) in each indicated group were harvested and washed triplicated in pre-cold PBS, cells were transferred into 500 µl Annexin V binding buffer with 5 µl of Annexin V-FITC and 5 µl (50 µg/mL) PI, continue to incubate for 15 minutes at room temperature (RT) in the dark. Flow cytometry analysis was applied to quantify the percentage of apoptosis of CRC cells in each indicated group. For the cell cycle analysis, after treatment, 5× 10 5 cells in each indicated group were harvested and washed in PBS, fixed in 70% ice clod ethanol at 4°C overnight, treated with RNaseA (50 µg/mL) and 0.1% Triton X-100, stained with PI (40 µg/ml) for 30 min at RT in the dark, followed by FACS analysis. For cell cycle distribution assessment, DNA content was determined using Mod fit LT software. The specific experimental procedures refer to the research of Tang et al. [ 21 ]. Xenograft mice assay 4–6 weeks old male BALB/c-nu/nu mice were purchased from Laboratory Animal Center of the Chongqing Medical University used for xenograft tumorigenicity assays. In this study, all mice were randomly allocated to each indicated group(N = 5), mice experiment was designed in a single-blind trial. Briefly, a final concentration of 2 × 10 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 (N = 5). Tumor size was monitored with calipers per week. All mice were sacrificed at 6th week after injection. Use the following formula to calculate the tumor volume: tumor volume (V, mm 3 ) = 0.5 × (larger diameter (mm)) × (smaller diameter (mm) 2 ). The procedure of animal experiments in this study was approved by the Animal Ethics Committee of Chongqing medical university. Glucose assay The glucose concentration in the culture supernatant was determined by using the Glucose assay kit-WST (Dojindo, Japan). Briefly, the standard curves were constructed with standard glucose solution (0 µM to 0.5 µM). The cells (5×10 5 cells/well) were seeded in a 6-well microplate and cultured 24 h at 37°C, after the incubation, 100 µL of the supernatant was transferred to a 1.5 ml microtube and diluted 40-fold with ddH2O (defined as sample solution). 50 µL sample solution with 50 µL working solution were added to a 96-well microplate. The assay plate was incubated at 37°C for 30 minutes. Then use a microplate reader to measure the absorbance of each well at 450 nm and the concentrations of glucose in each sample solution was calculated from the above standard curve. Flow cytometry and magnetic cell sorting 5×10 5 CRC cells in each indicated group were suspended in 200 µL cell staining buffer (BioLegend Cat. No. 420201) and labeled with 10 µL conjugated anti-human CD44-FITC (BioLegend Cat. No. 338804) or CD133-PE (BioLegend Cat. No.372804) for 15 min at 4°C in dark. Centrifuged at 350g for 5 minutes and washed twice with 2ml of cell staining buffer. Afterward, CD44 or CD133 expression was examined by flow cytometry. Fluorescence-activated cell sorting (FACS) of CD44 + /CD133 + double-positive cells and CD44 − /CD133 − double-negative HCT-116 cells was accomplished on a BD FACS ARIA II high-speed cell sorter according to manufacturer’s instructions. Chemical and chemotherapy drug treatment Add PI3K inhibitor named LY294002 (Sigma) to the cell culture medium at a final concentration of 10 µM, 24h prior. The chemoresistance of CRC cells to 5-FU or oxaliplatin (LOHP) was analyzed using the CCK-8 kit (Dojindo, Japan). Briefly, 5,000 cells in each indicated group were seeded into a 96-well plate, and the cells were incubated with different concentrations of 5-FU or LOHP for 72 h. After incubation, discard the medium and replace with fresh medium containing 100 µL CCK-8 solution. OD values were determined at 450 nm using a microplate reader (Sanyo, Japan). Co-immunoprecipitation assay Co-immunoprecipitation was carried out using the Dynabeads Protein G Immunoprecipitation Kit (Thermo Fisher Scientific), according to the manufacturer’s procedures. Ab-conjugated magnetic beads were generated by coupled 5µg anti-HIF-1α or TCF7L2 antibody with 50 µL (1.5 mg) magnetic beads, resuspended in 200 µL Ab Binding and washing Buffer, incubate with gentle rotation for 15 minutes at RT. Following incubation, 500 µL cell lysates containing the antigen were gently pipetting to resuspend the magnetic bead-Ab complex, further incubate with rotation overnight at 4°C to allow the antigen (Ag) to bind to the magnetic bead-Ab complex. Finally, the magnetic bead-Ab-Ag complex was eluted in 20 µL elution buffer, 20 µL eluted samples were mixed with 20 µL protein loading buffer, heat the mixture product at 95°C for 5 minutes further for SDS/PAGE western blotting analysis. Normal rabbit IgG (Cell signaling Technology) was used as a negative control antibody. This experimental procedure refers to the previous study [ 21 ]. Chromatin immunoprecipitation assay ChIP assays were performed according to the protocol of the Simple ChIP Plus Enzymatic Chromatin IP Kit (Agarose Beads; Cell signaling Technology). 5µg of HIF-1α antibody or negative control rabbit IgG was used to immunoprecipitation in ChIP reactions. The primers designed for ChIP assay are listed in Supplementary Table S1 . The complete experimental method refers to our previous research [ 21 ]. Statistical analysis All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, Inc) and SPSS version 22.0 (SPSS, Chicago, IL). The Student unpaired t-test was used for the comparison between two groups. One-way ANOVA analysis was performed in multiple groups comparisons. Correlation analyses between group were assessed using Spearman rank correlation. Categorical variables were assessed by using chi-square test (or Fisher exact test). Kaplan-Meier log-rank test was used to analyze the overall survival curve data. All data represent as mean ± SD. P < 0.05 was considered statistically significant. Results The expression of TCF7L2 is upregulated in CRC cell lines under hypoxia microenvironment Before identified the function role of TCF7L2 in CRC progression, we initially examined the mRNA and protein expression level of TCF7L2 in Caco-2, HCT-116, HT-29, LoVo, SW480, SW620 cells. HIEC-6 was selected as normal small intestine epithelium cell. The results showed that the mRNA level of TCF7L2 was remarkably up-regulated in CRC cell lines compared to HIEC-6, Caco-2 and HCT-116 cell lines were selected for further knock down experiments for their relative high expression of TCF7L2 (Fig. 1 . A). TCF7L2 protein expression levels in CRC cell lines were similar with the mRNA levels in western blotting analysis (Fig. 1 B). To further elucidate the biological roles of TCF7L2 in CRC cell lines. We established stable TCF7L2 knock down Caco-2 and HCT-116 cell lines with specific TCF7L2 shRNA (h) Lentiviral Particles. After transfection with TCF7L2-shRNA, the mRNA and protein expression of TCF7L2 were checked by RT-PCR and western-blotting analysis, respectively. The results showed that TCF7L2 knockdown cells had significantly lower levels of TCF7L2 mRNA and protein compared to negative control cells lines (Fig. 1 C, 1 D). Hypoxia is one of the most critical niches during solid tumor progression. Next, we elevated the expression of TCF7L2 under hypoxia in Caco-2 and HCT-116 cell lines. The results showed that under hypoxic conditions, the mRNA and protein levels of TCF7L2 in Caco-2 and HCT-116 cell lines increased significantly (Fig. 1 E, 1 F). TCF7L2 promotes CRC cells proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) in the presence of hypoxia The role of TCF7L2 in Caco-2 and HCT-116 cell lines in vitro proliferation was determined by CCK-8 analysis. The results indicated that the proliferation rates of Caco-2 and HCT-116 cell lines were significantly increased under hypoxic conditions. While deregulation of TCF7L2 block dramatically inhibited hypoxia-induced hyper-proliferation (Fig. 2 . A). Given that the TCF7L2 plays an important role in CRC cell proliferation in vitro, we next sought to investigate the impact of TCF7L2 on the tumorigenicity of CRC cells in vivo. As shown in the results, knockdown of TCF7L2 significantly suppress the tumor growth in vivo (Fig. 2 B, 2 C). Therefore, these results indicate that TCF7L2 plays a vital role in cancer progression both in vitro and in vivo. Glucose is essential for cell proliferation, growth, and survival. To test the effects of TCF7L2 on cancer metabolism, we conducted glucose assay which enables quantitation of glucose as a substrate in energy metabolism. We found that a decrease in the level of glucose in the cell suspension of Caco-2 and HCT-116 cell lines after hypoxia stimulation. Conversely, TCF7L2 knockdown in those cells, a reverse trend was detected (Fig. 2 D), suggesting that TCF7L2 may affect the efficiency of glucose utilization of CRC cells. Metastasis is considered as a crucial event during CRC development. To investigate the role of TCF7L2 in hypoxia induced CRC metastasis capability, trans-well migration and invasion assays were employed. As portrayed in the results, both Caco-2 and HCT-116 cell lines under hypoxia showed significantly enhanced cell migration and invasion capabilities. However, enforced knock down expression of TCF7L2 blocked Caco-2 and HCT-116 cell migration and invasion (Fig. 2 E, 2 F). Matrix metallopeptidases (MMPs) have been shown to play an important role in tumor invasion and metastasis. MMP-2 and MMP-9 are the critical MMPs regulators for cell migration and invasion. Under hypoxia, high expression of MMP-9 but not MMP-2 was observed in Caco-2 and HCT-116 cell lines. While knock-down TCF7L2 by shRNA decreased MMP-9 expression levels (Fig. 2 G). More and more evidence confirmed that hypoxia plays an important role in cancer metastasis and EMT. To further explore the molecular mechanism underlying the role of TCF7L2 in CRC development, we next detect the protein expression level of EMT markers and related transcription factors. As shown in the results, hypoxia significantly caused downregulation of epithelial marker E-cadherin but increased the mRNA expression level of mesenchymal markers N-cadherin, vimentin as well as transcriptional factors Snail and Slug. Conversely, TCF7L2 knockdown in Caco-2 and HCT116 cells under hypoxia led to an opposite expression pattern of these EMT related genes (Fig. 2 H). Consistently with the expression profiles of mRNA, western blotting assay confirmed that hypoxia stimulation significantly promoted EMT progression in CRC cells. While, enforced knockdown of TCF7L2 partially reversed the role of hypoxia in EMT activation of Caco-2 and HCT116 cells (Fig. 2 I). Collectively, these results indicate that TCF7L2 can stimulate the survival and metastatic ability of CRC cell lines. TCF7L2 involved in hypoxia induced apoptosis resistance and cell cycle arrest in CRC cell lines Apoptosis resistance is another common event and plays an essential role during tumor progression response to hypoxia, to further investigate the role of TCF7L2 in Caco-2 and HCT-116 cell lines under hypoxia, the apoptosis rate was detected. We observed that, under hypoxia conditions, the apoptosis rate of Caco-2 and HCT-116 cell lines were significantly decreased. While shRNA-mediated knockdown of TCF7L2 reduces hypoxia induced apoptosis resistance (Fig. 3 A). To further study the role of TCF7L2 in the survival of CRC cells, we examined the cell cycle progression in Caco-2 and HCT-116 cell lines by analyzing the distribution of cell cycle phrases. Our results showed that, the ratio of Caco-2 and HCT-116 cell lines in G0/G1 phrase was significantly decreased under hypoxia conditions. And the knockdown of TCF7L2 increases the proportion of cells in the G0/G1 phrase (Fig. 3 B). In addition, under hypoxia, the expression of cyclinD1 protein and the expression of proliferating cell nuclear antigen (PCNA) in Caco-2 and HCT-116 cell lines increased significantly. Conversely, down regulated expression level of cyclinD1 and PCNA were detected in TCF7L2 knock down cells (Fig. 3 C). Taking together, all results demonstrated that TCF7L2 promotes CRC cells survival by inducing apoptosis resistance and cell cycle G1/S transition. TCF7L2 promotes CRC cells proliferation though the PI3K/Akt signaling pathway Knowing that PI3K/Akt signaling pathway plays an important role in the proliferation of CRC cells, as shown in the results, the protein expression of p-PI3K p85 and p-AKT1(Ser 473) were significantly increased in Caco-2 and HCT116 cells under hypoxia. While knock down of TCF7L2 abrogated hypoxia induced PI3K/AKT signaling activation (Fig. 3 D). To further confirm that PI3K/Akt signaling pathway is involved in TCF7L2-induced cell proliferation, a well-known PI3K inhibitor LY294002(10µM, 24h) was pre-administered into stable TCF7L2 over-expressing Caco-2 and HCT116 cells to block PI3K/Akt signaling pathway. As shown in the results, activation of PI3K/AKT signaling was observed in stable TCF7L2 over-expressing Caco-2 and HCT116 cells. However, after LY294002 treated, almost completely blocked of p-PI3K p85 and p-AKT1(Ser 473) expression level was observed (Fig. 3 E). Moreover, over-expressing of TCF7L2 induced promote of cell proliferation could be partly reversed by inhibit PI3K in CRC cells (Fig. 3 F). Taken together, PI3K/Akt signaling pathway was essential for TCF7L2 mediated cell proliferation regulation in CRC cells. TCF7L2 involved in hypoxia induced chemo-resistance in the CRC cell lines Accumulating data has shown that hypoxia involved in chemo-resistance of various types of solid tumors due to the activation of HIF-1α signaling under hypoxia. To investigate whether TCF7L2 is involved in the chemical resistance of colorectal cancer cells induced by hypoxia in vitro, CRC cells were treated with gradient concentrations of 5-FU or oxaliplatin (LOHP), and then the cell survival rate was evaluated. The IC50 was then calculated in each group. For drug-induced apoptosis analysis, Caco-2 and HCT116 cells were pretreated with 5 µg/mL 5-FU or LOHP for 72h. As shown in the results, 5-FU or LOHP led to a dose-dependent decrease in the cell survival rate of Caco-2 and HCT116 cells. As expected, hypoxia stimulation significantly increased the resistance of Caco-2 and HCT116 cells to chemotherapeutic drugs (5-FU and LOHP), and the results showed that the IC50 of colorectal cancer cells cultured in hypoxia was significantly higher than their corresponding normoxia cells. Conversely, TCF7L2 knock down in Caco-2 and HCT116 cells abrogated hypoxia induced chemo-resistance (Fig. 4 A, 4 B, 4 C, 4 D). In addition, the results showed that hypoxia significantly reduced drug-induced apoptosis of CRC cells, while TCF7L2 knockdown showed the opposite effect (Fig. 4 E). These experimental data indicated that TCF7L2 is a potential regulator of chemo-sensitivity in CRC cells. TCF7L2 is associated with promoting CSC-like phenotype maintenance in CRC cells Given the potential role of TCF7L2 in promoting chemo-resistance in CRC cells prompted us to further explore whether TCF7L2 is crucial for of CRC cells. To address this, the colony formation and sphere formation assays were employed. As shown in the results, colony formation abilities of Caco-2 and HCT116 cells were enhanced under hypoxia, while significantly impaired following TCF7L2 knockdown (Fig. 5 A). As for tumor sphere assay, compare with HCT116 cells, Caco-2 cells were unable to form sphere in low-adherent, serum-free and growth factor supplied medium. As shown in the results, hypoxia led to significantly enhanced the sphere formation efficiency of HCT-116 cells. Interestingly, similarly finding were observed in the secondary passages. Conversely, TCF7L2 knock down significantly suppressed sphere formation and serially propagate of HCT-116 cells under hypoxia conditions (Fig. 5 B). Taking together, TCF7L2 plays a role in hypoxia regulating CSCs properties in CRC cells. To verify these observations, we next investigated the expression of some typical stemness-related markers by FACS (Fig. 5 C), RT-PCR (Fig. 5 D) and western-blotting (Fig. 5 E) in CRC cells. We found that some stemness related markers expression were significantly higher under hypoxia. While block TCF7L2 with TCF7L2-shRNA in CRC cells under hypoxia significantly decreased the expression of cancer stemness markers, including CD44, CD133, ALDH1A1, EpCAM, Nanog, OCT4. Thus, our results demonstrated that TCF7L2 may be served as a critical regulator of hypoxia derived stemness properties in CRC cells. Next, we explored to clarify that whether TCF7L2 was able to independently mediate the stemness of CRC caner stem-like cells. Cellular surface proteins CD44 and CD133 were known to identified as effective markers to isolate CSCs-like subpopulation from HCT-116 cells [ 22 ]. As shown in the results, CD44 + /CD133 + subpopulations displayed markedly enhanced sphere formation efficiency, while CD44 − /CD133 − subpopulations generated less sphere formation efficiency. As expected, TCF7L2 deletion in CD44 + /CD133 + subpopulations resulted in significantly downregulation the potentials of tumor sphere formation efficiency (Fig. 5 F). Next, flow cytometric assay (Fig. 5 G, 5 H) coupled with qRT-PCR (Fig. 5 I) and western-blotting (Fig. 5 J) assay were employed to further determine the effect of TCF7L2 on the expression of CSCs markers in CD44 + /CD133 + subpopulations. As shown in the results, TCF7L2 knock down had the capacities to decrease the expression abundance of these putative CSCs markers. Collectively, our finding demonstrated that TCF7L2 independently participates in cancer stem maintenance of CRC cells. Hypoxia induces the up-regulation of TCF7L2 through direct transcriptional induction by HIF-1α Hypoxia plays an important role in the development of CRC, our previous studies have shown that the expression of TCF7L2 mRNA and protein increases under hypoxia 21 . Numerously investigation indicated that HIF-1α and HIF-2α are the prominent regulators responsive to hypoxia in solid tumors. Next, we aim to clarify the molecular mechanism underlying hypoxia induced TCF7L2 expression in CRC cells, qRT-PCR and western-blotting methods were used to detect the mRNA and protein expression of HIF-1α and HIF-2α in Caco-2 and HCT116 cells under hypoxia conditions, the results showed that HIF-1α expression was significantly enhanced, while not HIF-2α. Consistently, under hypoxia, the mRNA expression of HIF-1α and HIF-1α-specific downstream target genes (including VEGF and GLUT1) in CRC cells was also significantly up-regulated (Fig. 6A, 6B). Next, Co-immunoprecipitation experiments confirmed that TCF7L2 does interact with HIF-1α in Caco-2 and HCT116 cells under hypoxia (Fig. 6C). Based on the above results, we asked whether HIF-1α can directly bind to the TCF7L2 promoter and regulate TCF7L2 gene transcription and expression. Online JASPAR analysis was employed and predicted the hypoxia responsive elements (HRE) potential binding site on TCF7L2 promoter region (Fig. 6D). Then ChIP assay analysis was then applied and confirmed that significant fold enrichment of HIF-1α binding with the HRE of TCF7L2 promoter was verified under hypoxia exposure in Caco-2 and HCT116 cells, as well as a considerable decrease in enrichment after HIF-1α knockdown (Fig. 6E). Finally, to investigate if HIF-1α plays an independent role in the observed increase expression of TCF7L2 induced by hypoxia. The Caco-2 and HCT-116 cells were transfection with HIF-1α shRNA to specifically knock down HIF-1α expression. As shown in the result, knock down of HIF-1α could significantly impaired hypoxia induced increased of TCF7L2 expression at protein and mRNA level (Fig. 6F, 6G). Taken together, our results demonstrated that hypoxia induced over expression of TCF7L2 in a HIF-1α dependent manner. Over expression of TCF7L2 and HIF-1αcorrelates with aberrant clinicopathological features in CRC patients To further investigate the clinical significance of TCF7L2 in CRC, we first quantified the TCF7L2 mRNA levels in 104 pairs of CRC specimens and adjacent normal colorectal tissues. We found that the expression level of TCF7L2 in colorectal cancer specimens was significantly higher than that of matched adjacent normal colorectal specimens (Fig. 7 A). Based on the expression profile of TCF7L2, we next explored the clinical significance of TCF7L2 in CRC patients. We divided 104 CRC patients into two groups based on TCF7L2 mRNA expression level. The results showed that TCF7L2 expression was positively correlated with T phase (p = 0.027) and metastasis (p = 0.011) (Table 1). Meanwhile, patients with high TCF7L2 expression levels showed significantly poorer overall survival (OS) rates (p = 0.0168) (Fig. 7 E). Next, we detected the mRNA expression level of HIF-1α in 104 pairs of CRC specimens and adjacent normal colorectal tissues, the results showed elevated levels of HIF-1α mRNA in CRC tissues (Fig. 7 B). Most importantly, a positive correlation between HIF-1α expression and TCF7L2 expression was observed in CRC specimens (Fig. 7 C). Consistently, the elevated protein expression level of HIF-1α and TCF7L2 were observed in 10 pairs of CRC specimens and adjacent normal colorectal tissues (Fig. 7 D). Based on the expression of HIF-1α and TCF7L2, CRC patients were further divided into following two groups: HIF-1α High TCF7L2 High group (n = 44) and HIF-1α Low TCF7L2 Low group (n = 26). The patients with high HIF-1a and TCF7L2 possessed aggressive clinicopathological feature including T stage (p = 0.02) and metastasis (p = 0.02) (Table 2). Most importantly, the prognosis of patients with high expression of TCF7L2 and HIF-1α is significantly worse than that of patients with low expression of TCF7L2 and HIF-1α (Fig. 7 F), showing that the combined use of TCF7L2 and HIF-1α can be served as an effective indicator to predict the prognosis of colorectal cancer patients. Discussion In the present study, we have elucidated the biological function and underlying mechanism of TCF7L2 in colorectal cancer (CRC). Our initial findings indicate that the overexpression of TCF7L2 under hypoxic conditions is implicated in cell proliferation, metastasis, and epithelial-mesenchymal transition (EMT) progression in CRC in vitro. Notably, TCF7L2 expression was found to be associated with the maintenance of cancer stemness in CRC cells. Mechanistically, we identified that TCF7L2 promotes CRC cell proliferation by activating the PI3K/AKT signaling pathway.Significantly, TCF7L2 has been identified as a key transcriptional regulator of HIF-1α, with hypoxia response element (HRE) binding sites located within the promoter region of HIF-1α, facilitating its transcriptional activation. In vivo studies revealed that TCF7L2 enhances tumor growth and metastasis in nude mice. Furthermore, our analysis demonstrated that the mRNA and protein expression levels of TCF7L2 in colorectal cancer (CRC) tissues are elevated compared to adjacent normal tissues, and this overexpression is associated with aberrant clinical features.Furthermore, colorectal cancer (CRC) patients exhibiting elevated expression levels of TCF7L2 and HIF-1α are associated with a poorer prognosis compared to those with lower expression levels of these markers. Increasing number of investigations indicated that TCF7L2, also known as Transcription factor 7-like 2, was elevated in carcinoma tissues and had been shown associated with poor prognosis [ 23 – 25 ]. Previously study reported that TCF7L2 protein was primarily localized in the cell nuclei of gastric cancer (GC) tissue, as well as in the cytoplasm in adjacent tissues. This suggested that TCF7L2 exerts a cancer-promoting role in the nucleus of GC cells and high TCF7L2 expression were significantly correlated with a poor prognosis for patients with GC. Functionally, they further indicated that TCF7L2 was found to be a major transcriptional regulator of PLAUR, with binding sites within the promoter region of urokinase-type plasminogen activator receptor (PLAUR), leading to its transcriptional activation, suggesting TCF7L2 play a vital role in regulating cell proliferation, anoikis resistance, and migration [ 26 ]. Xiang et.al reported that TCF7L2 positively regulated aerobic glycolysis by suppressing Egl-9 family hypoxia inducible factor 2 (EGLN2), leading to upregulation of hypoxia inducible factor 1 alpha subunit (HIF-1α), and TCF7L2 positively regulates HIF-1α stability and relevant glycolysis genes such as GLUT1, HK2, and LDHA in pancreatic cancer [ 27 ]. 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 named hypoxia inducible factors (HIFs). HIF-1α, as the predominant well-defined responsive regulator of hypoxic condition in solid tumors, regulates multiple target genes through various biological pathways [ 28 ]. Previous research reported that HIF-1α functions as a negative regulator of hARD1-mediated β-catenin acetylation, and under hypoxic conditions β-catenin is deacetylated due to HIF-1α competition with it for hARD1 binding, hARD1 is involved in the HIF-1α–mediated, hypoxic inactivation of TCF4 [ 29 ]. In the current study, we used ChIP analysis to identify TCF7L2 as an important downstream targeting gene for HIF-1α. Inconsistently with our finding, Kaidi and colleagues found that HIF-1α interacts with β-catenin via its NH2 terminal domain and that this interferes with the β-catenin–TCF7L2 association [ 30 ], suggesting a complex regulation network between HIF-1α and TCF7L2. EMT was a reversible process, which was initial studied during embryo morphogenesis. In addition, in recent years, it has been found that the state switching between EMT and MET plays a central role in various pathological processes, including tissue fibrosis, wound healing and early stages of cancer development [ 31 ]. More and more studies show that EMT is an early event of tumor metastasis, during EMT, cancer cells undergo phenotypic changes, epithelial cells transform into mesenchymal cells morphologically, resulting in enhanced cell motility and invasion ability. Epithelial cells have a typical apical–basal polarity structure, and the tight, adherent, and gap junctions between these cells limiting their ability to migrate and invasive. During EMT activation, epithelial cells lose cell polarity, lose cell-cell junctions, accompany with acquiring the ability of invade and migrate, transforming into mesenchymal cell morphology and characteristics [ 32 ]. Since EMT and hypoxic microenvironment in tumors may share multiple signaling pathways, recent studies have shown that hypoxia is an important factor leading to EMT-like phenotype changes in epithelial tumor cells [ 33 ]. Among all 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 reported that hypoxia enhancing migration ability, activating EMT and promoting MMPs expression in hepatocellular cancer cells by targeting AKT and HIF-1α/VEGF signaling pathway [ 34 ]. Grazia et.al indicated that overexpression of Pituitary adenylate cyclase-activating polypeptide (PACAP) was associated with hypoxia-induced EMT activation by regulating an important EMT-transcription factors (TFs), Zinc finger E-box-binding homeobox-1(ZEB1) in Glioblastoma [ 35 ]. Shi et.al also demonstrated PI3K/AKT signaling pathways were involved in hypoxia-induced EMT activation in colorectal cancer [ 36 ]. Coincidence with in previously results, our study demonstrated that under TME, both mRNA and protein expression of HIF-1α, TCF7L2 was upregulated in CRC cell lines Caco-2 and HCT116 cells, meanwhile, the migration and invasion capacities of CRC cells was dramatically enhanced after hypoxia stimulation, most importantly, epithelia marker E-cadherin was downregulated, mesenchymal markers Vimentin, N-cadherin and EMT-TFs snail, slug were significantly increased under TME. While knock down of TCF7L2 abrogated hypoxia induced EMT activation in CRC. Thus, we successfully demonstrate TCF7L2 is involved in hypoxia induced EMT progression of CRC. Accumulating evidence supports the idea that HIF-1α as an essential modulator for CSCs self-renewal and stemness traits maintenance in various carcinomas. CSC itself has a high degree of metabolic adaptability and can survive in an oxygen-deficient environment, while CSC's high acquisition and utilization for nutrients such as glucose enable them to survive in restricted glucose levels microenvironment, thereby promoting cell survival and tumorigenic potential [ 37 ]. We demonstrated that the colony and sphere formation abilities of CRC cells were remarkable enhanced in first and second passages under hypoxia, suggesting the important role of HIF-1α in CRC stemness maintenance. Moreover, in the current study, we observed that some typical stem genes such as CD44, CD133, ALDH1A1, EPCAM, NANOG, OCT4 were significantly enriched in CRC cells after hypoxia stimulation. On the contrary, downregulation of TCF7L2 exhibited the opposite effects. Both CD44 and CD133 also known as prominin-1 were known to be putative stem markers to isolate CSCs from CRC [ 38 ]. We then isolated CD44 + /CD133 + subpopulation (defined as CRC CSCs) to further explore whether TCF7L2 was involved in hypoxia facilitating the development of CRC through enhanced stemness of CRC CSCs. Our results demonstrated the independent role of TCF7L2 in cancer stemness maintenance of CRC. To date, the precise regulatory mechanism of TCF7L2 in colorectal cancer (CRC) remains unclear. In this study, we observed a significant association between TCF7L2 expression and the activation of the PI3K/AKT signaling pathway. The PI3K/AKT signaling pathway is known to play a crucial role in various biological processes, including cell proliferation, apoptosis, and cell cycle progression. Furthermore, this pathway has been reported to mediate the maintenance of stemness in various carcinomas, including liver cancer and colorectal cancer.In this study, we observed that TCF7L2 exerts a proliferative effect on colorectal cancer (CRC) cells by activating the PI3K/AKT signaling pathway.. Conclusions In conclusion, our current data have demonstrated that TCF7L2 plays a critical role in the progression of colorectal cancer (CRC). The overexpression of TCF7L2 was positively correlated with poor clinical features in CRC patients. Furthermore, we have elucidated a 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 finding provides a theoretical basis for considering TCF7L2 and HIF-1α as potential therapeutic targets for CRC. Declarations Conflict of interest The authors declare that they have no conflict of interest. Authors contribution K.T. and Y.C. designed and supervised the study; K.T. conducted the study; K.T. conducted the statistical analysis of experimental results; K.T. and Y.C. wrote and edited the paper; J.G., Y.L., and Y.C. reviewed the paper and approved the final version of the manuscript. Acknowledgement The authors would like to thank all the members of the laboratory. References Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin. 2024;74:12–49. 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. 2022;84:() 199–213. 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. 2010;57:9–16. Siegel R, DeSantis C, Virgo K, Stein K, Mariotto A, Smith T, et al. 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Trends Cell Biol. 2019;29:212–226. Davis FM, Stewart TA, Thompson EW, Monteith GR. Targeting EMT in cancer: opportunities for pharmacological intervention. Trends Pharmacol Sci. 2014;35:479–488. Hapke RY, Haake SM. Hypoxia-induced epithelial to mesenchymal transition in cancer. Cancer Lett. 2020;487:10–20. 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. 2019;33:798–807. 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. 2021;9:965. 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. 2021;11:3376–3391. 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. 2020;130:110623. 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. 2019;450:1–13. Tables Table 1 to 2 are available in the Supplementary Files section. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files Table.1.docx Table.2.docx SupplementaryTable1.docx SupplementaryTable2.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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4860804","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":345625436,"identity":"343b7fce-f15b-4f5e-8116-e0343c5ec8ec","order_by":0,"name":"Yong Cheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYBAC+/sH0j984LGpZ2xvIFbPDYZnjDNk0hKYew4QrYXxGTOPzeEE9hkJROpgnN2c9oAnJy2Pd+bjjTcYamyiCWphljmWbiBxxqZYcnZasQXDsbTcBkJa2BhyEiQMe9IYN87OMZNgbDhMWAsPQ/4HicR/hxn33zxDpBYJiYQ0iQM8hxMbZ/AQqcWA50CyYQNPmjFjD9AvCcT4xYC9IfHxHx4bOcb2wxtvfKixIawFRbtEAinKIVpI1TEKRsEoGAUjAwAA4HtDXmVdi2YAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-5652-7376","institution":"The First Affiliated Hospital of Chongqing Medical University, Chongqing, China.","correspondingAuthor":true,"prefix":"","firstName":"Yong","middleName":"","lastName":"Cheng","suffix":""},{"id":345625437,"identity":"f94b7310-47ba-49ad-bc9b-d4e236a21d4d","order_by":1,"name":"Kang Tang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kang","middleName":"","lastName":"Tang","suffix":""},{"id":345625438,"identity":"634443fa-34d0-4450-af85-18de8d06d94e","order_by":2,"name":"Jianping Gong","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jianping","middleName":"","lastName":"Gong","suffix":""},{"id":345625439,"identity":"892f3489-3b8a-41fa-bebf-27feb621dc9e","order_by":3,"name":"Yang Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-08-05 09:21:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4860804/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4860804/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":66853974,"identity":"229938c2-7ef4-4cd7-a1f6-6d0588118a1d","added_by":"auto","created_at":"2024-10-17 07:27:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1341684,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of TCF7L2 in CRC cell lines and stable TCF7L2 knockdown CRC cell lines.\u003c/strong\u003e \u003cstrong\u003eA, B\u003c/strong\u003e Using qRT-PCR and western-blotting methods to detect the expression of TCF7L2 mRNA and protein in different CRC cell lines, HIEC-6 was used as a human normal small intestine cell line, β-actin as a loading control (N=3). \u003cstrong\u003eC, D\u003c/strong\u003eUsing qRT-PCR and western-blotting analysis to confirm the knockdown efficiency of TCF7L2 shRNA (h) lentiviral particles in Caco-2 and HCT-116 cell lines (N=3). \u003cstrong\u003eE, F\u003c/strong\u003e The expression levels of TCF7L2 mRNA and protein in Caco-2 and HCT-116 cells under normoxia and hypoxic conditions were detected by qRT-PCR and western-blotting methods(N=3), β-actin as a loading control. Data are shown as mean ± SD, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4860804/v1/ec3fe14aafaca030bf16795c.png"},{"id":66853980,"identity":"c61471c9-1786-4dae-ab11-3e8240a3274c","added_by":"auto","created_at":"2024-10-17 07:27:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6855913,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of TCF7L2 in cell survival, migration, invasion and EMT in CRC cell lines. \u003c/strong\u003eA, Evaluation of the cell proliferation ability of designated groups of Caco-2 and HCT-116 cells in vitro by CCK-8 assay (N=3). \u003cstrong\u003eB, C\u003c/strong\u003e Photograph and growth curves of xenograft tumor in nude mice(N=5). \u003cstrong\u003eD\u003c/strong\u003e Measurement of glucose consumption levels in indicated CRC cells were determined by a glucose assay kit (N=3). \u003cstrong\u003eE, F\u003c/strong\u003e Representative pictures and statistic chart of migration and invasion in indicated Caco-2 and HCT-116 cells, respectively (N=3). G The protein expression levels of MMP-2 and MMP-9 were determined by Western blotting(N=3). \u003cstrong\u003eH, I \u003c/strong\u003eWestern blotting analysis and qRT-PCR of EMT markers in indicadted Caco-2 and HCT-116 cells (N=3), β-actin was used as loading control. Data are shown as mean ± SD, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4860804/v1/cad3001ba0318385f7b4275a.png"},{"id":66854166,"identity":"a9443993-8ae1-44cf-a79a-a862237df99b","added_by":"auto","created_at":"2024-10-17 07:35:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":373075,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTCF7L2 participated in hypoxia mediating chemo-resistance.\u003c/strong\u003e \u003cstrong\u003eA, C\u003c/strong\u003e Cell viability was quantified by the CCK-8 assay in indicated cells treated with concentration ranges of 5-FU or LOHP for 72h, respectively(N=3). \u003cstrong\u003eB, D\u003c/strong\u003e Quantify of resistance to 5-FU or LOHP in indicated cells was determined as the ratio of IC50 which was obtained from the inhibition curve(N=3). \u003cstrong\u003eE \u003c/strong\u003e5-FU or LOHP induced apoptosis in indicated cells was determined by FACS analysis (N=3). Data are shown as mean ± SD, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure46.png","url":"https://assets-eu.researchsquare.com/files/rs-4860804/v1/d7bfe816b037700229d53504.png"},{"id":66853977,"identity":"480da7f5-f5eb-4cb7-a749-9697f8766adb","added_by":"auto","created_at":"2024-10-17 07:27:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4557735,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of TCF7L2 on cell apoptosis and cell cycle in CRC cell lines. A \u003c/strong\u003eThe representative pictures and statistical bar chart for the cell apoptosis rate were determined by the FACS analysis using FITC Annexin V Apoptosis Detection Kit(N=3). \u003cstrong\u003eB\u003c/strong\u003eRepresentative cell-cycle FACS pictures and quantification of cell cycle phases in indicated CRC cells were analyzed by FACS analysis(N=3). \u003cstrong\u003eC\u003c/strong\u003e The protein expression level of cell cycle related genes CyclinD1 and PCNA were detected by western-blotting assay(N=3). \u003cstrong\u003eD\u003c/strong\u003e Protein expression of PI3K, p-PI3K, Akt, p-Akt in indicated cells was detected by Western blot analysis(N=3). \u003cstrong\u003eE\u003c/strong\u003e Protein expression analysis of TCF7L2, PI3K/AKT pathway activation and sensitivity to PI3K inhibition in indicated CRC cells(N=3). β-actin as a loading control (N=3). \u003cstrong\u003eF\u003c/strong\u003e TCF7L2 overexpressing CRC cells were treated with PI3K inhibitor LY294002, and cell proliferation ability was determined by CCK-8 assay(N=3). Data are shown as mean ± SD, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4860804/v1/cb3c668a3d424a6258ea9197.png"},{"id":66854167,"identity":"b667e5d8-98ce-48a5-a24a-1e26f6487442","added_by":"auto","created_at":"2024-10-17 07:35:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6057764,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe role of TCF7L2 in cancer stemness maintenance of CRC cell lines. A\u003c/strong\u003e Colony formation ability of CRC cells in indicated group was analyzed by colony formation assay(N=3). \u003cstrong\u003eB\u003c/strong\u003e Sphere formation assay was used to test the sphere formation abilities of indicated CRC cell lines. \u003cstrong\u003eC\u003c/strong\u003e FACS analysis of the expression of cell-surface markers CD44 and CD133 in indicated CRC cells(N=3). \u003cstrong\u003eD, E\u003c/strong\u003e The mRNA and protein expression level of CSCs-related markers in indicated CRC cells were determined by qRT-CPR and western blotting(N=3). \u003cstrong\u003eF \u003c/strong\u003eSphere formation ability of indicated sorting subpopulation was analyzed by sphere formation assay(N=3). \u003cstrong\u003eG, H\u003c/strong\u003e FACS of cell surface markers CD44 or CD133 expression in indicated CRC cells after sorting(N=3). \u003cstrong\u003eI, J\u003c/strong\u003e Detection of mRNA and protein expression levels of TCF7L2 and CSCs-related genes in indicated subpopulations by qRT-PCR and western blotting, and β-actin as a loading control (N=3). Data are shown as mean ± SD, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4860804/v1/f70e18f93d3ba71e34e2b4a7.png"},{"id":66853975,"identity":"688e5d4f-f0fa-487b-ae60-0b118a6e59ae","added_by":"auto","created_at":"2024-10-17 07:27:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3793080,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUp-regulation of TCF7L2 expression in CRC cells response to hypoxia depends on HIF-1α. A\u003c/strong\u003e The relative mRNA expression levels of TCF7L2, HIF-1α, HIF-2α, VEGF and GLUT1 in Caco-2 and HCT-116 cells in different periods of hypoxia culture were quantitatively detected by qRT-PCR(N=3). \u003cstrong\u003eB\u003c/strong\u003e Protein expression level of TCF7L2, HIF-1α, HIF-2α, VEGF and GLUT1 were determined by western blotting in Caco-2 and HCT-116 cells under different periods of hypoxia culture(N=3). \u003cstrong\u003eC\u003c/strong\u003e The endogenous interaction between HIF-1α and TCF7L2 in Caco-2 and HCT-116 cells was analyzed using Co-immunoprecipitation (co-IP) assay with the anti-HIF-1α or anti-TCF7L2 antibody, respectively. Immunoglobulin G (IgG) is used as the negative control(N=3). \u003cstrong\u003eD\u003c/strong\u003e HIF-1α binding motif (above), JASPAR online analysis predicted potential HIF-1α binding HRE within the TCF7L2 gene promoter region, promoter region defined as 1.0-kb upstream of the transcriptional start site (below). \u003cstrong\u003eE\u003c/strong\u003e ChIP 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). \u003cstrong\u003eF, G\u003c/strong\u003e qRT-PCR and western blotting assay demonstrated that hypoxia induced increased the mRNA and protein expression of TCF7L2 in Caco-2 and HCT-116 cells could be impaired by HIF-1a knock down(N=3). β-actin as a loading control (N=3). Data are shown as mean ± SD, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4860804/v1/bf94dd92d38e9cce9d9cf4ea.png"},{"id":66854164,"identity":"40acec24-9f37-48e0-b728-f0ca6fad0ee0","added_by":"auto","created_at":"2024-10-17 07:35:22","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1975917,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAberrantly elevated TCF7L2 and HIF-1α expression correlates with poor prognosis in colorectal cancer patients.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e TCF7L2 mRNA expression in 104 CRC tumor specimens and paired adjacent non-tumor tissues were analyzed by quantitative RT–PCR(N=104). \u003cstrong\u003eB\u003c/strong\u003e HIF-1α mRNA expression in 104 CRC tumor specimens and paired adjacent non-tumor tissues were analyzed by quantitative RT–PCR(N=104). \u003cstrong\u003eC\u003c/strong\u003e The correlation between the mRNA expression of TCF7L2 and HIF-1α in 104 CRC tumor tissues was tested with Spearman correlation analysis (N=104; r=0.6441; p\u0026lt;0.001). \u003cstrong\u003eD\u003c/strong\u003e TCF7L2 and HIF-1α protein expression in 10 CRC tumor specimens and paired adjacent non-tumor tissues were analyzed by western blotting. \u003cstrong\u003eE \u003c/strong\u003eKaplan–Meyer analysis of overall survival curves comparing TCF7L2 high(N=61) and low(N=43) expressing patients. \u003cstrong\u003eF\u003c/strong\u003e 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. β-actin as a loading control. Data are shown as mean ± SD, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4860804/v1/840098e61bd5180a28e2f95b.png"},{"id":68206066,"identity":"2133e69b-cad1-4fc9-835b-7bf0d49a3966","added_by":"auto","created_at":"2024-11-04 16:19:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4467407,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4860804/v1/798506a9-ca56-445c-bfaa-e142286ace20.pdf"},{"id":66853979,"identity":"31d9d2bd-271e-4fe0-a119-80899ac64973","added_by":"auto","created_at":"2024-10-17 07:27:22","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12649,"visible":true,"origin":"","legend":"","description":"","filename":"Table.1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4860804/v1/0494eecbeeaef1753d7989db.docx"},{"id":66854161,"identity":"09f8693f-77c0-43a8-b8c1-66ba6aa13bcb","added_by":"auto","created_at":"2024-10-17 07:35:21","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13095,"visible":true,"origin":"","legend":"","description":"","filename":"Table.2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4860804/v1/d72b6b040b4db6e4391e9cec.docx"},{"id":66853971,"identity":"3f1d09c8-5cd3-49b8-8b2d-08c98745e9e3","added_by":"auto","created_at":"2024-10-17 07:27:21","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":17383,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4860804/v1/fb537c3a0504ce62f5727061.docx"},{"id":66855638,"identity":"cd453113-d027-4f38-86b8-ee44aff1b51e","added_by":"auto","created_at":"2024-10-17 07:43:21","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":18257,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4860804/v1/b7a09bf8886ce4f54277ef87.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"TCF7L2 promotes tumor progression by regulating hypoxia-inducible factor 1 alpha through activating the PI3K/AKT signaling pathway in colorectal carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer (CRC) is among the most prevalent solid malignancies and represents the third leading cause of tumor-related mortality worldwide, with the third highest disease-specific death rate [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although multiple treatments such as whole genome sequencing (WGS) analysis, total mesorectal excision (TME), neoadjuvant chemotherapy or chemoradiotherapy and multidisciplinary team management (MDT) have been rapidly developed and widely applied in clinics for past decades [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Unfortunately, combination treatment including targeted therapy for advanced CRC has limited effectiveness, patients with advanced colorectal cancer still have poor survival, with 5-year survival rate is only about 12% [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, in recent years, mounting studies had focused on identifying the biological molecular mechanism underlying CRC initiation and metastasis. These urgently prompted us to explore novel effective molecular therapeutics targets to improve survival in patients with colorectal cancer.\u003c/p\u003e \u003cp\u003eHypoxia in one of the most common and critical microenvironments during solid tumors progression [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. It has been reported that hypoxia-inducible factors participate in proliferation, inflammation reaction, angiogenesis, invasion and distant metastasis in various types of solid carcinoma [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In response to the hypoxic cellular microenvironment, cancer cells undergo a complex series of adaptive changes including cell energy metabolism, proliferation, apoptosis, invasion and angiogenesis. Hypoxia inducible factor-1 (HIF-1) are predominant responsive regulator to hypoxia during tumor progression, which is a heterodimer consisting of α and β subunits. Many studies showed that in the presence of hypoxic microenvironment, HIF-1α evades the degradation by inhibition of the hydroxylation of PHD and combined with HIF-1β subunit to form heterodimer, translocate from the cytoplasm into the nucleus, they interact with specific DNA sequences contain hypoxia response elements (HRE), and mediates the transcription and expression of multiple downstream target genes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. To date, over 100 HIF downstream target genes have been discovered, most of these genes involved in the metastasis cascade, including epithelial\u0026ndash;mesenchymal transition (EMT), extracellular matrix (ECM), enhanced tumor cell motility, angiogenesis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Clinically, in most solid carcinoma types including breast cancer [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], hepatocellular carcinoma [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], ovarian cancer [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], esophageal cancer [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and colorectal cancer [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], elevated expression of HIF-1α was identified to positively associated with poor prognosis.\u003c/p\u003e \u003cp\u003eTCF7L2, also known as Transcription factor 7-like 2, which has been shown directly regulates genes involved in metabolism and cell cycle control within adipocytes by genome-wide analysis of TCF7L2 binding and gene expression [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Recently, TCF7L2 had been discovered expressing in various types of non-mineralizing soft cancerous tissues including colon, esophageal, lung, skin and stomach, suggesting TCF7L2 might play an essential role in the carcinogenesis of malignant tumors[\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It was reported that TCF7L2 gene was associated with type 2 diabetes, but it is not associated with cancer, except in reverse with prostate cancer, in nondiabetic participants, the association between TCF7L2 and colon cancer was still observed [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Moreover, a previously study indicated that TCF7L2 was found overexpression in mammary epithelial cell-derived organoids and involved in EMT, more importantly, cellular abnormal expression of TCF7L2 protein was positively associated with increased expression level of HIF-1α [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Similarity, in clear cell renal cell carcinoma (ccRCC), hypoxia microenvironment was reported to increase the expression of TCF7L2 in a HIF-2α dependent manner, and Hypoxia regulated TCF7L2 high expression also participated in ccRCC tumor survival and distant metastasis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, to the best of our knowledge, there are few investigations focus on the function of TCF7L2 in CRC.\u003c/p\u003e \u003cp\u003eOur study initially identified an elevated expression level of TCF7L2 in colorectal cancer (CRC) both in vivo and in vitro. Furthermore, overexpression of TCF7L2 in CRC patients was found to correlate with advanced clinical stages and metastasis. In addition, this research represents the first investigation into the relationship between TCF7L2 expression and CRC cell proliferation, survival, epithelial-mesenchymal transition (EMT), and the maintenance of cancer stemness. Finally, we explored the molecular mechanisms underlying the involvement of TCF7L2 in CRC progression.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eClinical specimens and cell culture\u003c/h2\u003e \u003cp\u003e104 Clinical CRC samples and adjacent non-tumor samples were gained from the Second Affiliated Hospital of Chongqing Medical University (Chongqing, China) from 2009\u0026ndash;2013. No patients received preoperative radiotherapy or chemotherapy before surgery. All patients in this study provided informed written consent and approved by the Ethical Committee of the Second Affiliated Hospital of Chongqing Medical University. CRC cell lines Caco-2, HCT-116, HT-29, LoVo, SW480, SW620 and the human immortalized normal small intestine epithelium cell lines HIEC-6 were obtained from the American Type Culture Collection (ATCC). All CRC cell lines were cultured in Leibovitz\u0026rsquo;s L-15 medium (Gibco, CA, USA) with 10% FBS (Thermo Fisher Scientific) at 37˚C and 5% CO\u003csub\u003e2\u003c/sub\u003e. HIEC-6 cell lines were cultured in Opti-MEM I Reduced Serum Medium (Gibco) supplied with 4% FBS (Thermo Fisher Scientific) and 10 ng/mL Epidermal Growth Factor (Sigma, St Louis, USA) at 37 ˚C and 5% CO\u003csub\u003e2\u003c/sub\u003e. To mimic the hypoxic microenvironment, cells were cultured in hypoxic cell incubator (Thermo Fisher Scientific) supplied 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 \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRNA interference\u003c/h2\u003e \u003cp\u003eThe TCF7L2 shRNA (h) Lentiviral Particles (Santa Cruz, SC-43525-V) or HIF-1α shRNA (h) Lentiviral Particles (Santa Cruz, sc-35561-V) were transfected in Caco-2 and HCT-116 cells to establish stable knock down of TCF7L2 or HIF-1α. Generally, CRC cell lines (5\u0026times;10\u003csup\u003e4\u003c/sup\u003e/well) were plated in a 6-well plate 24h prior to lentiviral particles transfection. Then the thawed lentiviral particles were transfected into cells with a multiplicity of infection (MOI\u0026thinsp;=\u0026thinsp;4) co-cultured with 5 \u0026micro;g/ml polybrene (Santa Cruz). Stable clones expressing the TCF7L2 or HIF-1α shRNA were selected by using 10 \u0026micro;g/ml Puromycin dihydrochloride (Sigma) for 14 days. Control shRNA Lentiviral Particles (Santa Cruz, sc-108080) were transduced into cells as a negative control. The TCF7L2 Lentiviral Activation Particles (h) (Santa Cruz, sc-400607-LAC) was transfected in Caco-2 and HCT-116 cells to establish stable over-expression of TCF7L2 cell lines. Control Lentiviral Activation Particles (Santa Cruz, sc-437282) were transduced into cells as a negative control. The specific operation procedure and method was referenced in previous studies [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and quantitative real-time PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA from tissues or cultured CRC cells was isolated with RNAiso plus (TaKaRa) according to the manufacture\u0026rsquo;s procedures. cDNA was synthesized with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The qRT-PCR analyses were conducted according to a previously described. The primer sequences designed for qRT-PCR analysis were listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. All reactions were performed in triplicate. The specific experimental methods were previously published [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eWestern-blot analysis\u003c/h2\u003e \u003cp\u003eProteins were extracted from frozen tissues or cultured CRC cells with phosphatase inhibitor contained RIPA solution (Sigma). 20 \u0026micro;g protein samples were subjected by SDS-PAGE gel and transferred onto a PVDF membrane. After blocked in 3% Bovine Serum Albumins (BSA) diluted with 0.1% TBST solution at room temperature for 1h. Then membranes were incubated at 4\u0026deg;C overnight with the primary Antibodies, the information of all primary antibodies used in this study is provided in Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. Subsequently, the membranes were incubated at room temperature for 1h with anti-rabbit IgG HRP secondary Ab (1:2000 dilution, sc‐2301; Santa Cruz) or anti‐mouse IgG HRP secondary Ab (1:2000 dilution, sc‐2005; Santa Cruz). The specific experimental steps refer to the previous study [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell proliferation, colony formation and sphere formation assay\u003c/h2\u003e \u003cp\u003eThe cell proliferation assay was carried out using the Cell Counting Kit-8 (CCK-8; Dojindo, Japan) at indicated time points according to the manufactures protocol. Colony-formation assays were performed as follows. Briefly, 1,000 cells/well were plated into a sex-well plate and cultured in complete cell medium for 12 days. The colonies in each group were then fixed and stained with Diff-Quick kit (Sysmex, Kobe, Japan). The colony number was only counted by containing at least of 50 cells/colony. Plating efficiency (%)\u0026thinsp;=\u0026thinsp;the number of colonies formed / the number of cells seeded \u0026times;100. For sphere formation assay, 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells were cultured on low-adherence plates in 10 mL serum-free medium with supplements of epidermal growth factor (EGF, 10 ng/ml), basic fibroblast growth factor (bFGF, 20 ng/ml), 4% B27 and 4 \u0026micro;g/ml insulin. Sphere formation efficiency (SFE, %)\u0026thinsp;=\u0026thinsp;the number of spheres formed at 14th day / the number of cells seeded \u0026times;100. The specific experimental methods were previously published [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTrans well migration and invasion assay\u003c/h2\u003e \u003cp\u003eIn vitro migration (without Matrigel pre-coated) assays and invasion (with Matrigel pre-coated) were performed using 24-well trans-well plates with 6.5 mm diameter and 8 \u0026micro;m pores filters (Corning). Briefly, CRC cells (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were plated into the upper chamber, trans-well plates were then incubated for 24h (migration) or 30h (invasion) at 37\u0026deg;C, respectively. Finally, migrated or invaded cells attached on the lower chamber were then gently washed with PBS, fixed and stained with Diff-Quick stain kit (Sysmex, Kobe, Japan). The procedure of the experiment was conducted by referring to previous studies [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell apoptosis and cell cycle analysis\u003c/h2\u003e \u003cp\u003eCell apoptosis analysis was performed by using the annexin V\u0026ndash;FITC Apoptosis Detection Kit (BD Biosciences) following the manufacturer\u0026rsquo;s protocol. Briefly, CRC cells (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells) in each indicated group were harvested and washed triplicated in pre-cold PBS, cells were transferred into 500 \u0026micro;l Annexin V binding buffer with 5 \u0026micro;l of Annexin V-FITC and 5 \u0026micro;l (50 \u0026micro;g/mL) PI, continue to incubate for 15 minutes at room temperature (RT) in the dark. Flow cytometry analysis was applied to quantify the percentage of apoptosis of CRC cells in each indicated group. For the cell cycle analysis, after treatment, 5\u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells in each indicated group were harvested and washed in PBS, fixed in 70% ice clod ethanol at 4\u0026deg;C overnight, treated with RNaseA (50 \u0026micro;g/mL) and 0.1% Triton X-100, stained with PI (40 \u0026micro;g/ml) for 30 min at RT in the dark, followed by FACS analysis. For cell cycle distribution assessment, DNA content was determined using Mod fit LT software. The specific experimental procedures refer to the research of Tang et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eXenograft mice assay\u003c/h2\u003e \u003cp\u003e4\u0026ndash;6 weeks old male BALB/c-nu/nu mice were purchased from Laboratory Animal Center of the Chongqing Medical University used for xenograft tumorigenicity assays. In this study, all mice were randomly allocated to each indicated group(N\u0026thinsp;=\u0026thinsp;5), mice experiment was designed in a single-blind trial. Briefly, a final concentration of 2 \u0026times; 10\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 (N\u0026thinsp;=\u0026thinsp;5). Tumor size was monitored with calipers per week. All mice were sacrificed at 6th week after injection. Use the following formula to calculate the tumor volume: tumor volume (V, mm\u003csup\u003e3\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;0.5 \u0026times; (larger diameter (mm)) \u0026times; (smaller diameter (mm)\u003csup\u003e2\u003c/sup\u003e). The procedure of animal experiments in this study was approved by the Animal Ethics Committee of Chongqing medical university.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGlucose assay\u003c/h2\u003e \u003cp\u003eThe glucose concentration in the culture supernatant was determined by using the Glucose assay kit-WST (Dojindo, Japan). Briefly, the standard curves were constructed with standard glucose solution (0 \u0026micro;M to 0.5 \u0026micro;M). The cells (5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well) were seeded in a 6-well microplate and cultured 24 h at 37\u0026deg;C, after the incubation, 100 \u0026micro;L of the supernatant was transferred to a 1.5 ml microtube and diluted 40-fold with ddH2O (defined as sample solution). 50 \u0026micro;L sample solution with 50 \u0026micro;L working solution were added to a 96-well microplate. The assay plate was incubated at 37\u0026deg;C for 30 minutes. Then use a microplate reader to measure the absorbance of each well at 450 nm and the concentrations of glucose in each sample solution was calculated from the above standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry and magnetic cell sorting\u003c/h2\u003e \u003cp\u003e5\u0026times;10\u003csup\u003e5\u003c/sup\u003e CRC cells in each indicated group were suspended in 200 \u0026micro;L cell staining buffer (BioLegend Cat. No. 420201) and labeled with 10 \u0026micro;L conjugated anti-human CD44-FITC (BioLegend Cat. No. 338804) or CD133-PE (BioLegend Cat. No.372804) for 15 min at 4\u0026deg;C in dark. Centrifuged at 350g for 5 minutes and washed twice with 2ml of cell staining buffer. Afterward, CD44 or CD133 expression was examined by flow cytometry. Fluorescence-activated cell sorting (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\u003edouble-negative HCT-116 cells was accomplished on a BD FACS ARIA II high-speed cell sorter according to manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eChemical and chemotherapy drug treatment\u003c/h2\u003e \u003cp\u003eAdd PI3K inhibitor named LY294002 (Sigma) to the cell culture medium at a final concentration of 10 \u0026micro;M, 24h prior. The chemoresistance of CRC cells to 5-FU or oxaliplatin (LOHP) was analyzed using the CCK-8 kit (Dojindo, Japan). Briefly, 5,000 cells in each indicated group were seeded into a 96-well plate, and the cells were incubated with different concentrations of 5-FU or LOHP for 72 h. After incubation, discard the medium and replace with fresh medium containing 100 \u0026micro;L CCK-8 solution. OD values were determined at 450 nm using a microplate reader (Sanyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCo-immunoprecipitation assay\u003c/h2\u003e \u003cp\u003eCo-immunoprecipitation was carried out using the Dynabeads Protein G Immunoprecipitation Kit (Thermo Fisher Scientific), according to the manufacturer\u0026rsquo;s procedures. Ab-conjugated magnetic beads were generated by coupled 5\u0026micro;g anti-HIF-1α or TCF7L2 antibody with 50 \u0026micro;L (1.5 mg) magnetic beads, resuspended in 200 \u0026micro;L Ab Binding and washing Buffer, incubate with gentle rotation for 15 minutes at RT. Following incubation, 500 \u0026micro;L cell lysates containing the antigen were gently pipetting to resuspend the magnetic bead-Ab complex, further incubate with rotation overnight at 4\u0026deg;C to allow the antigen (Ag) to bind to the magnetic bead-Ab complex. Finally, the magnetic bead-Ab-Ag complex was eluted in 20 \u0026micro;L elution buffer, 20 \u0026micro;L eluted samples were mixed with 20 \u0026micro;L protein loading buffer, heat the mixture product at 95\u0026deg;C for 5 minutes further for SDS/PAGE western blotting analysis. Normal rabbit IgG (Cell signaling Technology) was used as a negative control antibody. This experimental procedure refers to the previous study [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eChromatin immunoprecipitation assay\u003c/h2\u003e \u003cp\u003eChIP assays were performed according to the protocol of the Simple ChIP Plus Enzymatic Chromatin IP Kit (Agarose Beads; Cell signaling Technology). 5\u0026micro;g of HIF-1α antibody or negative control rabbit IgG was used to immunoprecipitation in ChIP reactions. The primers designed for ChIP assay are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The complete experimental method refers to our previous research [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, Inc) and SPSS version 22.0 (SPSS, Chicago, IL). The Student unpaired t-test was used for the comparison between two groups. One-way ANOVA analysis was performed in multiple groups comparisons. Correlation analyses between group were assessed using Spearman rank correlation. Categorical variables were assessed by using chi-square test (or Fisher exact test). Kaplan-Meier log-rank test was used to analyze the overall survival curve data. All data represent as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eThe expression of TCF7L2 is upregulated in CRC cell lines under hypoxia microenvironment\u003c/h2\u003e \u003cp\u003eBefore identified the function role of TCF7L2 in CRC progression, we initially examined the mRNA and protein expression level of TCF7L2 in Caco-2, HCT-116, HT-29, LoVo, SW480, SW620 cells. HIEC-6 was selected as normal small intestine epithelium cell. The results showed that the mRNA level of TCF7L2 was remarkably up-regulated in CRC cell lines compared to HIEC-6, Caco-2 and HCT-116 cell lines were selected for further knock down experiments for their relative high expression of TCF7L2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. A). TCF7L2 protein expression levels in CRC cell lines were similar with the mRNA levels in western blotting analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). To further elucidate the biological roles of TCF7L2 in CRC cell lines. We established stable TCF7L2 knock down Caco-2 and HCT-116 cell lines with specific TCF7L2 shRNA (h) Lentiviral Particles. After transfection with TCF7L2-shRNA, the mRNA and protein expression of TCF7L2 were checked by RT-PCR and western-blotting analysis, respectively. The results showed that TCF7L2 knockdown cells had significantly lower levels of TCF7L2 mRNA and protein compared to negative control cells lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Hypoxia is one of the most critical niches during solid tumor progression. Next, we elevated the expression of TCF7L2 under hypoxia in Caco-2 and HCT-116 cell lines. The results showed that under hypoxic conditions, the mRNA and protein levels of TCF7L2 in Caco-2 and HCT-116 cell lines increased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTCF7L2 promotes CRC cells proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) in the presence of hypoxia\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe role of TCF7L2 in Caco-2 and HCT-116 cell lines in vitro proliferation was determined by CCK-8 analysis. The results indicated that the proliferation rates of Caco-2 and HCT-116 cell lines were significantly increased under hypoxic conditions. While deregulation of TCF7L2 block dramatically inhibited hypoxia-induced hyper-proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. A). Given that the TCF7L2 plays an important role in CRC cell proliferation in vitro, we next sought to investigate the impact of TCF7L2 on the tumorigenicity of CRC cells in vivo. As shown in the results, knockdown of TCF7L2 significantly suppress the tumor growth in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Therefore, these results indicate that TCF7L2 plays a vital role in cancer progression both in vitro and in vivo.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGlucose is essential for cell proliferation, growth, and survival. To test the effects of TCF7L2 on cancer metabolism, we conducted glucose assay which enables quantitation of glucose as a substrate in energy metabolism. We found that a decrease in the level of glucose in the cell suspension of Caco-2 and HCT-116 cell lines after hypoxia stimulation. Conversely, TCF7L2 knockdown in those cells, a reverse trend was detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), suggesting that TCF7L2 may affect the efficiency of glucose utilization of CRC cells.\u003c/p\u003e \u003cp\u003eMetastasis is considered as a crucial event during CRC development. To investigate the role of TCF7L2 in hypoxia induced CRC metastasis capability, trans-well migration and invasion assays were employed. As portrayed in the results, both Caco-2 and HCT-116 cell lines under hypoxia showed significantly enhanced cell migration and invasion capabilities. However, enforced knock down expression of TCF7L2 blocked Caco-2 and HCT-116 cell migration and invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Matrix metallopeptidases (MMPs) have been shown to play an important role in tumor invasion and metastasis. MMP-2 and MMP-9 are the critical MMPs regulators for cell migration and invasion. Under hypoxia, high expression of MMP-9 but not MMP-2 was observed in Caco-2 and HCT-116 cell lines. While knock-down TCF7L2 by shRNA decreased MMP-9 expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eMore and more evidence confirmed that hypoxia plays an important role in cancer metastasis and EMT. To further explore the molecular mechanism underlying the role of TCF7L2 in CRC development, we next detect the protein expression level of EMT markers and related transcription factors. As shown in the results, hypoxia significantly caused downregulation of epithelial marker E-cadherin but increased the mRNA expression level of mesenchymal markers N-cadherin, vimentin as well as transcriptional factors Snail and Slug. Conversely, TCF7L2 knockdown in Caco-2 and HCT116 cells under hypoxia led to an opposite expression pattern of these EMT related genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Consistently with the expression profiles of mRNA, western blotting assay confirmed that hypoxia stimulation significantly promoted EMT progression in CRC cells. While, enforced knockdown of TCF7L2 partially reversed the role of hypoxia in EMT activation of Caco-2 and HCT116 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Collectively, these results indicate that TCF7L2 can stimulate the survival and metastatic ability of CRC cell lines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eTCF7L2 involved in hypoxia induced apoptosis resistance and cell cycle arrest in CRC cell lines\u003c/h2\u003e \u003cp\u003eApoptosis resistance is another common event and plays an essential role during tumor progression response to hypoxia, to further investigate the role of TCF7L2 in Caco-2 and HCT-116 cell lines under hypoxia, the apoptosis rate was detected. We observed that, under hypoxia conditions, the apoptosis rate of Caco-2 and HCT-116 cell lines were significantly decreased. While shRNA-mediated knockdown of TCF7L2 reduces hypoxia induced apoptosis resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further study the role of TCF7L2 in the survival of CRC cells, we examined the cell cycle progression in Caco-2 and HCT-116 cell lines by analyzing the distribution of cell cycle phrases. Our results showed that, the ratio of Caco-2 and HCT-116 cell lines in G0/G1 phrase was significantly decreased under hypoxia conditions. And the knockdown of TCF7L2 increases the proportion of cells in the G0/G1 phrase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In addition, under hypoxia, the expression of cyclinD1 protein and the expression of proliferating cell nuclear antigen (PCNA) in Caco-2 and HCT-116 cell lines increased significantly. Conversely, down regulated expression level of cyclinD1 and PCNA were detected in TCF7L2 knock down cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Taking together, all results demonstrated that TCF7L2 promotes CRC cells survival by inducing apoptosis resistance and cell cycle G1/S transition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eTCF7L2 promotes CRC cells proliferation though the PI3K/Akt signaling pathway\u003c/h2\u003e \u003cp\u003eKnowing that PI3K/Akt signaling pathway plays an important role in the proliferation of CRC cells, as shown in the results, the protein expression of p-PI3K p85 and p-AKT1(Ser 473) were significantly increased in Caco-2 and HCT116 cells under hypoxia. While knock down of TCF7L2 abrogated hypoxia induced PI3K/AKT signaling activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). To further confirm that PI3K/Akt signaling pathway is involved in TCF7L2-induced cell proliferation, a well-known PI3K inhibitor LY294002(10\u0026micro;M, 24h) was pre-administered into stable TCF7L2 over-expressing Caco-2 and HCT116 cells to block PI3K/Akt signaling pathway. As shown in the results, activation of PI3K/AKT signaling was observed in stable TCF7L2 over-expressing Caco-2 and HCT116 cells. However, after LY294002 treated, almost completely blocked of p-PI3K p85 and p-AKT1(Ser 473) expression level was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Moreover, over-expressing of TCF7L2 induced promote of cell proliferation could be partly reversed by inhibit PI3K in CRC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Taken together, PI3K/Akt signaling pathway was essential for TCF7L2 mediated cell proliferation regulation in CRC cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eTCF7L2 involved in hypoxia induced chemo-resistance in the CRC cell lines\u003c/h2\u003e \u003cp\u003eAccumulating data has shown that hypoxia involved in chemo-resistance of various types of solid tumors due to the activation of HIF-1α signaling under hypoxia. To investigate whether TCF7L2 is involved in the chemical resistance of colorectal cancer cells induced by hypoxia in vitro, CRC cells were treated with gradient concentrations of 5-FU or oxaliplatin (LOHP), and then the cell survival rate was evaluated. The IC50 was then calculated in each group. For drug-induced apoptosis analysis, Caco-2 and HCT116 cells were pretreated with 5 \u0026micro;g/mL 5-FU or LOHP for 72h. As shown in the results, 5-FU or LOHP led to a dose-dependent decrease in the cell survival rate of Caco-2 and HCT116 cells. As expected, hypoxia stimulation significantly increased the resistance of Caco-2 and HCT116 cells to chemotherapeutic drugs (5-FU and LOHP), and the results showed that the IC50 of colorectal cancer cells cultured in hypoxia was significantly higher than their corresponding normoxia cells. Conversely, TCF7L2 knock down in Caco-2 and HCT116 cells abrogated hypoxia induced chemo-resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In addition, the results showed that hypoxia significantly reduced drug-induced apoptosis of CRC cells, while TCF7L2 knockdown showed the opposite effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). These experimental data indicated that TCF7L2 is a potential regulator of chemo-sensitivity in CRC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eTCF7L2 is associated with promoting CSC-like phenotype maintenance in CRC cells\u003c/h2\u003e \u003cp\u003eGiven the potential role of TCF7L2 in promoting chemo-resistance in CRC cells prompted us to further explore whether TCF7L2 is crucial for of CRC cells. To address this, the colony formation and sphere formation assays were employed. As shown in the results, colony formation abilities of Caco-2 and HCT116 cells were enhanced under hypoxia, while significantly impaired following TCF7L2 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). As for tumor sphere assay, compare with HCT116 cells, Caco-2 cells were unable to form sphere in low-adherent, serum-free and growth factor supplied medium. As shown in the results, hypoxia led to significantly enhanced the sphere formation efficiency of HCT-116 cells. Interestingly, similarly finding were observed in the secondary passages. Conversely, TCF7L2 knock down significantly suppressed sphere formation and serially propagate of HCT-116 cells under hypoxia conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Taking together, TCF7L2 plays a role in hypoxia regulating CSCs properties in CRC cells. To verify these observations, we next investigated the expression of some typical stemness-related markers by FACS (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), RT-PCR (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 CRC cells. We found that some stemness related markers expression were significantly higher under hypoxia. While block TCF7L2 with TCF7L2-shRNA in CRC cells under hypoxia significantly decreased the expression of cancer stemness markers, including CD44, CD133, ALDH1A1, EpCAM, Nanog, OCT4. Thus, our results demonstrated that TCF7L2 may be served as a critical regulator of hypoxia derived stemness properties in CRC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we explored to clarify that whether TCF7L2 was able to independently mediate the stemness of CRC caner stem-like cells. Cellular surface proteins CD44 and CD133 were known to identified as effective markers to isolate CSCs-like subpopulation from HCT-116 cells [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. As shown in the results, CD44\u003csup\u003e+\u003c/sup\u003e/CD133\u003csup\u003e+\u003c/sup\u003e subpopulations displayed markedly enhanced sphere formation efficiency, while CD44\u003csup\u003e\u0026minus;\u003c/sup\u003e/CD133\u003csup\u003e\u0026minus;\u003c/sup\u003e subpopulations generated less sphere formation efficiency. As expected, TCF7L2 deletion in CD44\u003csup\u003e+\u003c/sup\u003e/CD133\u003csup\u003e+\u003c/sup\u003e subpopulations resulted in significantly downregulation the potentials of tumor sphere formation efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Next, flow cytometric assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH) coupled with qRT-PCR (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) assay were employed to further determine the effect of TCF7L2 on the expression of CSCs markers in CD44\u003csup\u003e+\u003c/sup\u003e/CD133\u003csup\u003e+\u003c/sup\u003e subpopulations. As shown in the results, TCF7L2 knock down had the capacities to decrease the expression abundance of these putative CSCs markers. Collectively, our finding demonstrated that TCF7L2 independently participates in cancer stem maintenance of CRC cells.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eHypoxia induces the up-regulation of TCF7L2 through direct transcriptional induction by HIF-1α\u003c/h2\u003e \u003cp\u003eHypoxia plays an important role in the development of CRC, our previous studies have shown that the expression of TCF7L2 mRNA and protein increases under hypoxia\u003csup\u003e21\u003c/sup\u003e. Numerously investigation indicated that HIF-1α and HIF-2α are the prominent regulators responsive to hypoxia in solid tumors. Next, we aim to clarify the molecular mechanism underlying hypoxia induced TCF7L2 expression in CRC cells, qRT-PCR and western-blotting methods were used to detect the mRNA and protein expression of HIF-1α and HIF-2α in Caco-2 and HCT116 cells under hypoxia conditions, the results showed that HIF-1α expression was significantly enhanced, while not HIF-2α. Consistently, under hypoxia, the mRNA expression of HIF-1α and HIF-1α-specific downstream target genes (including VEGF and GLUT1) in CRC cells was also significantly up-regulated (Fig.\u0026nbsp;6A, 6B). Next, Co-immunoprecipitation experiments confirmed that TCF7L2 does interact with HIF-1α in Caco-2 and HCT116 cells under hypoxia (Fig.\u0026nbsp;6C). Based on the above results, we asked whether HIF-1α can directly bind to the TCF7L2 promoter and regulate TCF7L2 gene transcription and expression. Online JASPAR analysis was employed and predicted the hypoxia responsive elements (HRE) potential binding site on TCF7L2 promoter region (Fig.\u0026nbsp;6D). Then ChIP assay analysis was then applied and confirmed that significant fold enrichment of HIF-1α binding with the HRE of TCF7L2 promoter was verified under hypoxia exposure in Caco-2 and HCT116 cells, as well as a considerable decrease in enrichment after HIF-1α knockdown (Fig.\u0026nbsp;6E). Finally, to investigate if HIF-1α plays an independent role in the observed increase expression of TCF7L2 induced by hypoxia. The Caco-2 and HCT-116 cells were transfection with HIF-1α shRNA to specifically knock down HIF-1α expression. As shown in the result, knock down of HIF-1α could significantly impaired hypoxia induced increased of TCF7L2 expression at protein and mRNA level (Fig.\u0026nbsp;6F, 6G). Taken together, our results demonstrated that hypoxia induced over expression of TCF7L2 in a HIF-1α dependent manner.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eOver expression of TCF7L2 and HIF-1αcorrelates with aberrant clinicopathological features in CRC patients\u003c/h2\u003e \u003cp\u003eTo further investigate the clinical significance of TCF7L2 in CRC, we first quantified the TCF7L2 mRNA levels in 104 pairs of CRC specimens and adjacent normal colorectal tissues. We found that the expression level of TCF7L2 in colorectal cancer specimens was significantly higher than that of matched adjacent normal colorectal specimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Based on the expression profile of TCF7L2, we next explored the clinical significance of TCF7L2 in CRC patients. We divided 104 CRC patients into two groups based on TCF7L2 mRNA expression level. The results showed that TCF7L2 expression was positively correlated with T phase (p\u0026thinsp;=\u0026thinsp;0.027) and metastasis (p\u0026thinsp;=\u0026thinsp;0.011) (Table\u0026nbsp;1). Meanwhile, patients with high TCF7L2 expression levels showed significantly poorer overall survival (OS) rates (p\u0026thinsp;=\u0026thinsp;0.0168) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Next, we detected the mRNA expression level of HIF-1α in 104 pairs of CRC specimens and adjacent normal colorectal tissues, the results showed elevated levels of HIF-1α mRNA in CRC tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Most importantly, a positive correlation between HIF-1α expression and TCF7L2 expression was observed in CRC specimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Consistently, the elevated protein expression level of HIF-1α and TCF7L2 were observed in 10 pairs of CRC specimens and adjacent normal colorectal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Based on the expression of HIF-1α and TCF7L2, CRC patients were further divided into following two groups: HIF-1α\u003csup\u003eHigh\u003c/sup\u003e TCF7L2\u003csup\u003eHigh\u003c/sup\u003e group (n\u0026thinsp;=\u0026thinsp;44) and HIF-1α\u003csup\u003eLow\u003c/sup\u003e TCF7L2\u003csup\u003eLow\u003c/sup\u003e group (n\u0026thinsp;=\u0026thinsp;26). The patients with high HIF-1a and TCF7L2 possessed aggressive clinicopathological feature including T stage (p\u0026thinsp;=\u0026thinsp;0.02) and metastasis (p\u0026thinsp;=\u0026thinsp;0.02) (Table\u0026nbsp;2). Most importantly, the prognosis of patients with high expression of TCF7L2 and HIF-1α is significantly worse than that of patients with low expression of TCF7L2 and HIF-1α (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eF), showing that the combined use of TCF7L2 and HIF-1α can be served as an effective indicator to predict the prognosis of colorectal cancer patients.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we have elucidated the biological function and underlying mechanism of TCF7L2 in colorectal cancer (CRC). Our initial findings indicate that the overexpression of TCF7L2 under hypoxic conditions is implicated in cell proliferation, metastasis, and epithelial-mesenchymal transition (EMT) progression in CRC in vitro. Notably, TCF7L2 expression was found to be associated with the maintenance of cancer stemness in CRC cells. Mechanistically, we identified that TCF7L2 promotes CRC cell proliferation by activating the PI3K/AKT signaling pathway.Significantly, TCF7L2 has been identified as a key transcriptional regulator of HIF-1α, with hypoxia response element (HRE) binding sites located within the promoter region of HIF-1α, facilitating its transcriptional activation. In vivo studies revealed that TCF7L2 enhances tumor growth and metastasis in nude mice. Furthermore, our analysis demonstrated that the mRNA and protein expression levels of TCF7L2 in colorectal cancer (CRC) tissues are elevated compared to adjacent normal tissues, and this overexpression is associated with aberrant clinical features.Furthermore, colorectal cancer (CRC) patients exhibiting elevated expression levels of TCF7L2 and HIF-1α are associated with a poorer prognosis compared to those with lower expression levels of these markers.\u003c/p\u003e \u003cp\u003eIncreasing number of investigations indicated that TCF7L2, also known as Transcription factor 7-like 2, was elevated in carcinoma tissues and had been shown associated with poor prognosis [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Previously study reported that TCF7L2 protein was primarily localized in the cell nuclei of gastric cancer (GC) tissue, as well as in the cytoplasm in adjacent tissues. This suggested that TCF7L2 exerts a cancer-promoting role in the nucleus of GC cells and high TCF7L2 expression were significantly correlated with a poor prognosis for patients with GC. Functionally, they further indicated that TCF7L2 was found to be a major transcriptional regulator of PLAUR, with binding sites within the promoter region of urokinase-type plasminogen activator receptor (PLAUR), leading to its transcriptional activation, suggesting TCF7L2 play a vital role in regulating cell proliferation, anoikis resistance, and migration [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Xiang et.al reported that TCF7L2 positively regulated aerobic glycolysis by suppressing Egl-9 family hypoxia inducible factor 2 (EGLN2), leading to upregulation of hypoxia inducible factor 1 alpha subunit (HIF-1α), and TCF7L2 positively regulates HIF-1α stability and relevant glycolysis genes such as GLUT1, HK2, and LDHA in pancreatic cancer [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\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 named hypoxia inducible factors (HIFs). HIF-1α, as the predominant well-defined responsive regulator of hypoxic condition in solid tumors, regulates multiple target genes through various biological pathways [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Previous research reported that HIF-1α functions as a negative regulator of hARD1-mediated β-catenin acetylation, and under hypoxic conditions β-catenin is deacetylated due to HIF-1α competition with it for hARD1 binding, hARD1 is involved in the HIF-1α\u0026ndash;mediated, hypoxic inactivation of TCF4 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In the current study, we used ChIP analysis to identify TCF7L2 as an important downstream targeting gene for HIF-1α. Inconsistently with our finding, Kaidi and colleagues found that HIF-1α interacts with β-catenin via its NH2 terminal domain and that this interferes with the β-catenin\u0026ndash;TCF7L2 association [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], suggesting a complex regulation network between HIF-1α and TCF7L2.\u003c/p\u003e \u003cp\u003eEMT was a reversible process, which was initial studied during embryo morphogenesis. In addition, in recent years, it has been found that the state switching between EMT and MET plays a central role in various pathological processes, including tissue fibrosis, wound healing and early stages of cancer development [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. More and more studies show that EMT is an early event of tumor metastasis, during EMT, cancer cells undergo phenotypic changes, epithelial cells transform into mesenchymal cells morphologically, resulting in enhanced cell motility and invasion ability. Epithelial cells have a typical apical\u0026ndash;basal polarity structure, and the tight, adherent, and gap junctions between these cells limiting their ability to migrate and invasive. During EMT activation, epithelial cells lose cell polarity, lose cell-cell junctions, accompany with acquiring the ability of invade and migrate, transforming into mesenchymal cell morphology and characteristics [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Since EMT and hypoxic microenvironment in tumors may share multiple signaling pathways, recent studies have shown that hypoxia is an important factor leading to EMT-like phenotype changes in epithelial tumor cells [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Among all 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 reported that hypoxia enhancing migration ability, activating EMT and promoting MMPs expression in hepatocellular cancer cells by targeting AKT and HIF-1α/VEGF signaling pathway [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Grazia et.al indicated that overexpression of Pituitary adenylate cyclase-activating polypeptide (PACAP) was associated with hypoxia-induced EMT activation by regulating an important EMT-transcription factors (TFs), Zinc finger E-box-binding homeobox-1(ZEB1) in Glioblastoma [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Shi et.al also demonstrated PI3K/AKT signaling pathways were involved in hypoxia-induced EMT activation in colorectal cancer [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Coincidence with in previously results, our study demonstrated that under TME, both mRNA and protein expression of HIF-1α, TCF7L2 was upregulated in CRC cell lines Caco-2 and HCT116 cells, meanwhile, the migration and invasion capacities of CRC cells was dramatically enhanced after hypoxia stimulation, most importantly, epithelia marker E-cadherin was downregulated, mesenchymal markers Vimentin, N-cadherin and EMT-TFs snail, slug were significantly increased under TME. While knock down of TCF7L2 abrogated hypoxia induced EMT activation in CRC. Thus, we successfully demonstrate TCF7L2 is involved in hypoxia induced EMT progression of CRC.\u003c/p\u003e \u003cp\u003eAccumulating evidence supports the idea that HIF-1α as an essential modulator for CSCs self-renewal and stemness traits maintenance in various carcinomas. CSC itself has a high degree of metabolic adaptability and can survive in an oxygen-deficient environment, while CSC's high acquisition and utilization for nutrients such as glucose enable them to survive in restricted glucose levels microenvironment, thereby promoting cell survival and tumorigenic potential [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. We demonstrated that the colony and sphere formation abilities of CRC cells were remarkable enhanced in first and second passages under hypoxia, suggesting the important role of HIF-1α in CRC stemness maintenance. Moreover, in the current study, we observed that some typical stem genes such as CD44, CD133, ALDH1A1, EPCAM, NANOG, OCT4 were significantly enriched in CRC cells after hypoxia stimulation. On the contrary, downregulation of TCF7L2 exhibited the opposite effects. Both CD44 and CD133 also known as prominin-1 were known to be putative stem markers to isolate CSCs from CRC [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. We then isolated CD44\u003csup\u003e+\u003c/sup\u003e/CD133\u003csup\u003e+\u003c/sup\u003e subpopulation (defined as CRC CSCs) to further explore whether TCF7L2 was involved in hypoxia facilitating the development of CRC through enhanced stemness of CRC CSCs. Our results demonstrated the independent role of TCF7L2 in cancer stemness maintenance of CRC.\u003c/p\u003e \u003cp\u003eTo date, the precise regulatory mechanism of TCF7L2 in colorectal cancer (CRC) remains unclear. In this study, we observed a significant association between TCF7L2 expression and the activation of the PI3K/AKT signaling pathway. The PI3K/AKT signaling pathway is known to play a crucial role in various biological processes, including cell proliferation, apoptosis, and cell cycle progression. Furthermore, this pathway has been reported to mediate the maintenance of stemness in various carcinomas, including liver cancer and colorectal cancer.In this study, we observed that TCF7L2 exerts a proliferative effect on colorectal cancer (CRC) cells by activating the PI3K/AKT signaling pathway..\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, our current data have demonstrated that TCF7L2 plays a critical role in the progression of colorectal cancer (CRC). The overexpression of TCF7L2 was positively correlated with poor clinical features in CRC patients. Furthermore, we have elucidated a 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 finding provides a theoretical basis for considering TCF7L2 and HIF-1α as potential therapeutic targets for CRC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e \u003ch2\u003eAuthors contribution\u003c/h2\u003e \u003cp\u003eK.T. and Y.C. designed and supervised the study; K.T. conducted the study; K.T. conducted the statistical analysis of experimental results; K.T. and Y.C. wrote and edited the paper; J.G., Y.L., and Y.C. reviewed the paper and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThe authors would like to thank all the members of the laboratory.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. 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Effect of PACAP on Hypoxia-Induced Angiogenesis and Epithelial-Mesenchymal Transition in Glioblastoma. Biomedicines. 2021;9:965.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\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. 2021;11:3376\u0026ndash;3391.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\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. 2020;130:110623.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\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. 2019;450:1\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 2 are available in the Supplementary Files section.\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-4860804/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4860804/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHypoxia is a critical pathogenic factor in cancer development and metastasis. The pivotal role of hypoxia-inducible factor 1α (HIF-1α) in tumor progression under hypoxic conditions is well-documented. However, the specific mechanisms by which HIF-1α contributes to colorectal cancer (CRC) progression remain inadequately elucidated. In this study, we observed an upregulation of Transcription Factor 7-like 2 (TCF7L2) in CRC cells under hypoxic conditions. Meanwhile, hypoxia-induced overexpression of TCF7L2 plays a pivotal role in the proliferation, apoptosis, cell cycle arrest, migration, invasion, epithelial-mesenchymal transition (EMT), and cancer stem cell (CSC) characteristics of colorectal cancer (CRC) cells in vitro. Additionally, our findings indicate that the inhibition of TCF7L2 results in a significant reduction of tumor growth in vivo. Mechanistically, hypoxia-induced up-regulation of TCF7L2 expression occurs in a HIF-1α-dependent manner. Chromatin immunoprecipitation (ChIP) assays demonstrated increased HIF-1α binding to the promoter sequence of TCF7L2 following hypoxic stimulation. Furthermore, our findings indicate that TCF7L2 plays an oncogenic role in colorectal cancer (CRC) by activating the PI3K/AKT signaling pathway. Additionally, we observed that elevated expression levels of both HIF-1α and TCF7L2 in CRC specimens are associated with aberrant clinicopathological features. Co-expression of TCF7L2 and HIF-1α predicts a poor prognosis in CRC patients. Targeting TCF7L2 is a promising approach to colorectal cancer therapy.\u003c/p\u003e","manuscriptTitle":"TCF7L2 promotes tumor progression by regulating hypoxia-inducible factor 1 alpha through activating the PI3K/AKT signaling pathway in colorectal carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-17 07:27:16","doi":"10.21203/rs.3.rs-4860804/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":"f576a0ca-1f7d-48fb-816c-8381fd037eaf","owner":[],"postedDate":"October 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":36636419,"name":"Biological sciences/Cancer/Gastrointestinal cancer/Colorectal cancer"},{"id":36636420,"name":"Health sciences/Biomarkers/Prognostic markers"}],"tags":[],"updatedAt":"2024-11-04T16:11:06+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-17 07:27:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4860804","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4860804","identity":"rs-4860804","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}