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

doi:10.21203/rs.3.rs-4860804/v1

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

https://doi.org/10.21203/rs.3.rs-4860804/v1

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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

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.

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% CO₂. 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% CO₂. To mimic the hypoxic microenvironment, cells were cultured in hypoxic cell incubator (Thermo Fisher Scientific) supplied with 1% O₂, 5% CO₂ and 94% N₂.

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 (5×10⁴/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 × 10⁴ 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 × 10⁵) 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 × 10⁵ 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× 10⁵ 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 × 10⁶ 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, mm³) = 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 (5×10⁵ 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

5×10⁵ 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.

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. 1B). 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. 1C, 1D). 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. 1E, 1F).

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. 2B, 2C). 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. 2D), 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. 2E, 2F). 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. 2G).

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. 2H). 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. 2I). 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. 3A).

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. 3B). 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. 3C). 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. 3D). 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. 3E). Moreover, over-expressing of TCF7L2 induced promote of cell proliferation could be partly reversed by inhibit PI3K in CRC cells (Fig. 3F). 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. 4A, 4B, 4C, 4D). In addition, the results showed that hypoxia significantly reduced drug-induced apoptosis of CRC cells, while TCF7L2 knockdown showed the opposite effect (Fig. 4E). 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. 5A). 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. 5B). 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. 5C), RT-PCR (Fig. 5D) and western-blotting (Fig. 5E) 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. 5F). Next, flow cytometric assay (Fig. 5G, 5H) coupled with qRT-PCR (Fig. 5I) and western-blotting (Fig. 5J) 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 hypoxia²¹. 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. 7A). 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. 7E). 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. 7B). Most importantly, a positive correlation between HIF-1α expression and TCF7L2 expression was observed in CRC specimens (Fig. 7C). 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. 7D). 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. 7F), 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.

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..

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.

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.

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Table 1 to 2 are available in the Supplementary Files section.

There is NO conflict of interest to disclose.

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

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Abstract

Figures

Introduction

Materials and methods

Clinical specimens and cell culture

RNA interference

RNA extraction and quantitative real-time PCR (qRT-PCR)

Western-blot analysis

Cell proliferation, colony formation and sphere formation assay

Trans well migration and invasion assay

Cell apoptosis and cell cycle analysis

Xenograft mice assay

Glucose assay

Flow cytometry and magnetic cell sorting

Chemical and chemotherapy drug treatment

Co-immunoprecipitation assay

Chromatin immunoprecipitation assay

Statistical analysis

Results

The expression of TCF7L2 is upregulated in CRC cell lines under hypoxia microenvironment

TCF7L2 involved in hypoxia induced apoptosis resistance and cell cycle arrest in CRC cell lines

TCF7L2 promotes CRC cells proliferation though the PI3K/Akt signaling pathway

TCF7L2 involved in hypoxia induced chemo-resistance in the CRC cell lines

TCF7L2 is associated with promoting CSC-like phenotype maintenance in CRC cells

Hypoxia induces the up-regulation of TCF7L2 through direct transcriptional induction by HIF-1α

Over expression of TCF7L2 and HIF-1αcorrelates with aberrant clinicopathological features in CRC patients

Discussion

Conclusions

Declarations

Conflict of interest

Authors contribution

Acknowledgement

References

Tables

Additional Declarations

Supplementary Files

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