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YAP1 activates SLC2A1 transcription and augments the malignant behavior of colorectal cancer cells by activating the Wnt/β-catenin signaling pathway

Cell Division volume 20, Article number: 8 (2025) Cite this article

Abstract

Objective

This paper examined the role of solute carrier family 2 member 1 (SLC2A1) in colorectal cancer (CRC) progression, focusing on its expression levels, functional implications, and regulatory mechanisms involving Yes-associated protein 1 (YAP1) and the Wnt signaling pathway.

Methods

GEO datasets (GSE14297, GSE18462, GSE40367) were analyzed to identify genes linked to metastasis in CRC, and TCGA-COAD system was used to analyze the expression pattern and prognostic values of SLC2A1 in CRC. Functional studies were conducted using CRC cell lines (Caco-2 and SW480). Cell viability, migration and invasion, and apoptosis were examined using EdU assays, Transwell assays, and flow cytometry. YAP1’s regulatory role on SLC2A1 was investigated using ChIP-qPCR and luciferase reporter assays. The Wnt/β-catenin agonist SKL2001 was used for functional rescue experiments.

Results

SLC2A1 was upregulated in CRC cells, and its upregulation was associated with tumor metastasis and unfavorable outcomes according to bioinformatics. Knockdown of SLC2A1 resulted in reduced cell viability, decreased migration, and increased apoptosis in Caco-2 and SW480 cells. Additionally, YAP1 was identified as a transcriptional activator of SLC2A1. Knockdown of YAP1 decreased SLC2A1 expression and reduced expression of Wnt target genes, thus suppressing malignant behavior of tumor cells. However, further overexpression of SLC2A1 restored cell viability and migration in YAP1-deficient cells. The YAP1- SLC2A1 axis activated the Wnt/β-catenin by reducing GSK3β activity.

Conclusion

SLC2A1 is critical in CRC progression, with YAP1 serving as a key regulator of its expression and function. The YAP1-SLC2A1-Wnt axis represents a potential therapeutic target for CRC, providing insights into metabolic adaptations that support tumor growth and metastasis.

Introduction

Colorectal cancer (CRC) is one of the most prevalent cancers globally and leading cause of cancer-related mortality. The complex pathophysiology of CRC involves a combination of genetic, epigenetic, and environmental factors that contribute to tumor initiation, progression, and metastasis. Among these, altered metabolic processes and specific gene expressions are crucial in determining the disease’s aggressiveness and clinical outcomes [1,2,3].

A key player in cancer metabolism is the solute carrier family 2 member 1 (SLC2A1), also known as glucose transporter 1 (GLUT1). SLC2A1 facilitates the uptake of glucose into cells, supporting the elevated metabolic demands of rapidly proliferating tumor cells. Its expression is often upregulated in various cancers, including CRC, and is associated with poor prognosis and advanced disease stages [4,5,6,7]. Increased SLC2A1 levels not only support tumor growth but also impact the tumor microenvironment, influencing cancer cell behavior and their interactions with surrounding stromal cells [8, 9]. Studies have underscored the significance of SLC2A1in multiple cancer types. For instance, in breast cancer, overexpression of SLC2A1 correlates with enhanced glycolytic activity and a more aggressive tumor phenotype [10]. In glioblastoma, SLC2A1 expression is linked to the maintenance of cancer stem cells, contributing to tumor recurrence and treatment resistance [11, 12]. In ovarian cancer, SLC2A1 promotes cell proliferation and survival under hypoxic conditions [13, 14]. Similarly, in pancreatic cancer, high SLC2A1 expression facilitates tumor growth and is associated with poor patient outcomes [15, 16]. Targeting SLC2A1 in hepatocellular carcinoma has been shown to significantly inhibit tumor growth and enhance the drug efficacy [16, 17].

The transcriptional regulation of SLC2A1 is influenced by various signaling pathways, particularly the Hippo pathway and its effector, yes-associated protein 1 (YAP1). YAP1 promotes cell proliferation and survival and has been implicated in several malignancies, including CRC [18]. YAP1 has been demonstrated to enhance the expression of SLC2A1, augmenting its role in metabolic regulation that favors tumor growth [19]. In breast cancer, YAP1 activation is associated with increased glycolysis through upregulation of SLC2A1, driving tumor progression [20, 21]. Additionally, YAP1 has been shown to maintain stem cell properties in lung cancer by regulating metabolic pathways [22, 23]. Studies also suggest that YAP1 overexpression in melanoma enhances tumor growth, indicating a metabolic shift toward glycolysis [24, 25]. In pancreatic cancer, YAP1 promotes metabolic adaptation and resistance to chemotherapy, primarily through upregulation of SLC2A1 and other glycolytic enzymes [26, 27].

The Wnt signaling pathway, frequently dysregulated activated in cancer, exerts pivotal roles in tumorigenesis. Activation of Wnt signaling triggers nuclear translocation of β-catenin (CTNNB1), which modulates various target genes implicated in cell proliferation and migration [28]. In hepatocellular carcinoma, aberrant Wnt signaling is linked to increased SLC2A1 expression, facilitating tumor growth [29]. Wnt pathway activation has also been shown in gastric cancer, where it drives tumor aggressiveness [30, 31]. Furthermore, studies in triple-negative breast cancer reveal that Wnt signaling, through β-catenin, enhances glucose uptake, contributing to metabolic adaptation in the tumor microenvironment [32, 33]. In ovarian cancer, Wnt pathway activation is linked with poor survival, and its role in promoting glycolysis [34]. This pathway also plays a critical pathogenic role in CRC, making it a promising target for CRC management [4].

Despite the well-established roles of SLC2A1, YAP1, and Wnt signaling in CRC, the precise mechanisms connecting these factors to disease progression and metastasis remain poorly understood. This gap in knowledge hinders the development of targeted therapies that could improve patient outcomes. By elucidating the role of SLC2A1 in CRC and its interaction with YAP1 and Wnt signaling, this paper offers aims to advance our understanding of CRC progression.

Results

SLC2A1is upregulated in CRC and linked to metastasis and poor patient prognosis

We first downloaded the GSE14297, GSE18462, and GSE40367 datasets from the GEO database. Using screening thresholds of|Log FC > 1.0| and adjusted p-value < 0.05, DEGs associated with CRC metastasis were identified (Fig. 1A–C). Through intersection analysis, we identified the SLC2A1 gene (Fig. 1D). Further analysis of the TCGA-COAD database revealed that SLC2A1 expression was significantly higher in CRC tissues compared to normal colon tissues from the GTEx database (Fig. 1E). Additionally, elevated SLC2A1 expression correlated with poor outcomes in COAD patients, with higher levels associated with more advanced clinical stages (Fig. 1F–G). We also examined SLC2A1 expression in CRC cell lines (Caco-2, HCT116, and SW480) and the immortalized colonic mucosal cell line FHC, observing a marked increase in SLC2A1 levels in the CRC cell lines (Fig. 1H–I).

Fig. 1
figure 1

Elevated SLC2A1 levels in CRC.A–C Volcano plots for DEGs in GSE14297, GSE18462, and GSE40367 datasets; D Venn diagram of intersecting DEGs from GSE14297, GSE18462, and GSE40367; E Comparison of SLC2A1 levels in TCGA-COAD and normal colon tissues from the GTEx database; F Kaplan-Meier analysis of SLC2A1 levels in relation to patient survival in the TCGA-COAD database, Cut-off = SLC2A1 median; G SLC2A1 levels in patients with different clinical stages in the TCGA-COAD database; H, I qPCR and WB analyses of SLC2A1 levels in COAD cell lines Caco-2, HCT116, SW480, and immortalized colonic mucosal cell line FHC. Cell experiments were repeated 3–5 times, with statistical analysis performed using One-Way ANOVA, **P < 0.01

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SLC2A1 knockdown inhibits growth and mobility of CRC cells

Based on the results shown in Fig. 1H–I, we selected the two cell lines with the highest SLC2A1 expression, Caco-2 and SW480, for functional validation experiments. These cells were transfected with two different shRNAs targeting SLC2A1, and transfection efficiency was confirmed via qPCR and WB analysis (Fig. 2A–B). After SLC2A1 knockdown, both Caco-2 and SW480 cells showed a significant reduction in cell viability (Fig. 2C), as evidenced by a marked decrease in EdU-positive cells (Fig. 2D) and a substantial increase in apoptotic cell counts (Fig. 2E). Transwell assays revealed that SLC2A1 knockdown led to significantly reduced cell migration and invasion (Fig. 2F–G).

Fig. 2
figure 2

SLC2A1 knockdown inhibits growth and mobility of CRC cells.A, B qPCR and WB analyses of SLC2A1 levels in SW480 and Caco-2 cells; C CCK-8 assay assessing changes in cell viability; D EdU staining demonstrating proliferation capabilities; E Annexin-V/PI flow cytometry analysis detecting apoptosis rates; F, G Transwell assays evaluating migration and invasion abilities. Cell experiments were repeated 3–5 times, with statistical analysis performed using Two-Way ANOVA, **P < 0.01

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YAP1 transcriptionally activates SLC2A1

To investigate the upstream regulatory mechanisms of SLC2A1, we used the UCSC Genome Browser and JASPAR to predict transcription factors that could bind to the SLC2A1 promoter. Our analysis revealed a significant binding affinity between YAP1 and the SLC2A1 promoter (Fig. 3A–B). Next, luciferase reporter vectors containing the SLC2A1 promoter sequence were co-transfected with YAP1 overexpression vectors into HEK293T cells. We observed a marked increase in luciferase activity upon YAP1 overexpression (Fig. 3C), indicating that YAP1 binds to the SLC2A1 promoter. This interaction was further confirmed by anti-YAP1 ChIP-qPCR experiments (Fig. 3D). YAP1 levels were significantly elevated in CRC cell lines (Fig. 3E–F), and TCGA-COAD data also demonstrated increased YAP1 expression (Fig. 3G), which positively correlated with SLC2A1 levels (Fig. 3H). Additionally, following YAP1 knockdown in SW480 or Caco-2 cells, SLC2A1 levels were reduced (Fig. 3I–J).

Fig. 3
figure 3

YAP1 transcriptionally activates SLC2A1.A–B Predicted binding results of YAP1 to the SLC2A1 promoter from the JASPAR database and YAP1’s conserved binding sequence; C Co-transfection of the luciferase reporter vector containing the SLC2A1 promoter sequence and YAP1 overexpression vector in HEK293T cells, measuring luciferase activity; D ChIP assay using anti-YAP1, followed by qPCR analysis of SLC2A1 promoter levels in the precipitate; E, F qPCR and WB analyses of YAP1 levels in COAD cell lines Caco-2, HCT116, SW480, and the immortalized colonic mucosal cell line FHC; G Comparison of YAP1 levels in TCGA-COAD and normal colon tissues from the GTEx database; H Correlation between YAP1 and SLC2A1 levels in the TCGA-COAD database; I, J qPCR and WB analyses of YAP1 and SLC2A1 levels in SW480 and Caco-2 cells

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Overexpression of SLC2A1 restores malignant phenotype of CRC cells reduced by shYAP1

Given that the results in Fig. 3I–J indicated that YAP1 knockdown reduced SLC2A1 expression, we proceeded to overexpress SLC2A1 in cells with low YAP1 expression. Transfection efficiency was confirmed by qPCR and WB analyses (Fig. 4A–B). Notably, SLC2A1 overexpression significantly enhanced the growth and proliferative capacity of SW480 and Caco-2 cells (Fig. 4C–D), while the number of apoptotic cells sharply decreased (Fig. 4E). Furthermore, SLC2A1 overexpression promoted the migratory and invasive capabilities of SW480 and Caco-2 cells (Fig. 4F–G).

Fig. 4
figure 4

Overexpression of SLC2A1 restores malignant phenotype of CRC cells reduced by shYAP1.A, B qPCR and WB analyses of SLC2A1 levels in SW480 and Caco-2 cells; C CCK-8 assay assessing changes in cell viability; D EdU staining to evaluate the proliferative capacity; E Annexin-V/PI flow cytometry to assess the apoptotic ratio; F, G Transwell assays to evaluate the migratory and invasive capabilities. Cell experiments were repeated three to five times, and statistical analysis was performed using Two-Way ANOVA, **P < 0.01

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SLC2A1 activates the Wnt pathway and promotes nuclear translocation of CTNNB1

We next investigated Wnt signaling activation in Caco-2 and SW480 cells. YAP1 knockdown significantly reduced the levels of c-Myc, c-JUN, and c-Met. However, SLC2A1 overexpression restored these levels (Fig. 5A–B). Additionally, analysis of the TCGA-COAD dataset revealed significant positive correlations between c-Myc, c-JUN, c-Met, and SLC2A1 (Fig. 5C–E). Wnt signaling activation leads to CTNNB1 nuclear translocation. We assessed CTNNB1 nuclear localization via immunofluorescence and found that YAP1 knockdown significantly reduced its nuclear accumulation. In contrast, SLC2A1 overexpression reactivated the Wnt pathway (Fig. 5F). Taken together, our findings suggest that YAP1 promotes SLC2A1 expression, which activates the Wnt pathway, facilitates CTNNB1 nuclear translocation, and ultimately enhances CRC growth and metastasis.

Fig. 5
figure 5

SLC2A1 activates the Wnt pathway and promotes nuclear translocation of CTNNB1.A, B qPCR and WB analyses of c-Myc, c-JUN, and c-Met levels in Caco-2 and SW480 cells; C–E Correlation analysis of c-Myc, c-JUN, and c-Met levels with SLC2A1 in the TCGA-COAD dataset; F Immunofluorescence detection of CTNNB1 nuclear translocation in Caco-2 and SW480 cells. Cell experiments were repeated three to five times, and statistical analysis was performed using Two-Way ANOVA, **P < 0.01

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The YAP1-SLC2A1 axis activates the Wnt/β-catenin signaling pathway and augments CRC progression by reducing GSK3β activity

The findings above suggest that YAP1 knockdown led to a reduction in the nuclear translocation of CTNNB1, which was restored by the overexpression of SLC2A1. However, the exact mechanism by which SLC2A1 influences the nuclear translocation of CTNNB1 has not been thoroughly explored. Therefore, we first examined the expression levels of GSK3β and CTNNB1 in the cells. We found that after YAP1 knockdown in SW480 and Caco-2 cells, GSK3β expression slightly increased, while CTNNB1 expression decreased and phos-CTNNB1 expression increased. However, upon overexpressing SLC2A1, phosphorylation of GSK3β and CTNNB1 in cells was reduced, and CTNNB1 expression was restored (Fig. 6A).

Fig. 6
figure 6

The YAP1-SLC2A1 axis activates the Wnt/β-catenin signaling pathway and augments CRC progression by reducing GSK3β activity.A WB analyses of GSK3β, CTNNB1, and phos-CTNNB1 protein levels in SW480 and Caco-2 cells; B Activation of the Wnt/β-catenin signaling pathway in SW480 and Caco-2 cells determined using TOP/FOP luciferase reporter assays. SW480 and Caco-2 cells with stable SLC2A1 knockdown were treated with the Wnt agonist SKL2001. C Activation of the Wnt/β-catenin signaling pathway in SW480 and Caco-2 cells after SKL2001 treatment determined using TOP/FOP luciferase reporter assays; D CCK-8 assay to assess changes in cell viability; E EdU staining to evaluate the proliferative capacity. Cell experiments were repeated 3–5 times, with statistical analysis performed using Two-Way ANOVA, **P < 0.01

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Next, we assessed the activity of the TOP/LEF reporter in the cells. As anticipated, the YAP1 knockdown suppressed Wnt pathway activation, which was restored by SLC2A1 overexpression (Fig. 6B). To further confirm whether SLC2A1 modulates CRC cell growth by regulating Wnt/β-catenin activity, we treated SLC2A1-silenced cells with the Wnt/β-catenin agonist SKL2001, which stabilizes β-catenin by disrupting the Axin/β-catenin interaction. Importantly, following SKL2001 treatment, a significant increase was observed in TOP/FOP luciferase activity (Fig. 6C). This treatment also significantly increased growth and proliferation of the CRC cells (Fig. 6D–E).

Discussion

This research explored the functional significance of SLC2A1 in CRC and examined the regulatory mechanisms involving YAP1 and the Wnt pathway. Our findings confirmed elevated SLC2A1 levels in CRC patients, and SLC2A1 knockdown significantly inhibited cell growth, migration, and invasion in CRC cell lines. Additionally, we identified YAP1 as a key transcriptional regulator of SLC2A1, linking metabolic dysregulation to Wnt pathway activation, which plays critical roles in CRC progression.

Our results clearly underscore the significant role of SLC2A1 in CRC. The initial bioinformatics insights suggested that increased SLC2A1 expression in CRC tissues was associated with advanced clinical stages and poor patient prognosis, which aligns with existing literature that links GLUT1 overexpression to aggressive cancer phenotypes. For instance, Macheda et al. reported similar findings in breast cancer, where high GLUT1 expression correlated with increased glycolytic activity and poorer patient outcomes [35]. Yang et al. also supported this idea, demonstrating that SLC2A1 promotes cellular proliferation and survival in hypoxic conditions, which are common in solid tumors, thus reinforcing the metabolic adaptation of cancer cells [36]. Additionally, Serbulea et al. highlighted that SLC2A1 contributes to the tumor microenvironment by modulating lactate production, emphasizing its dual role in metabolic support and tumor progression [37].

Functional assays conducted in this study demonstrated that SLC2A1 knockdown in Caco-2 and SW480 cell lines significantly reduced cell viability and increased apoptosis. These findings are consistent with those of Zhang et al., who showed that GLUT1 inhibition in glioblastoma cells led to reduced cell growth and increased sensitivity to chemotherapeutic agents [38]. Additionally, our results revealed a marked decrease in EdU-positive cells and increased apoptotic markers, suggesting that SLC2A1 is not only essential for energy metabolism but also plays a critical role in maintaining cellular homeostasis in CRC. The observed reduction in migration and invasion following SLC2A1 knockdown further supports previous studies highlighting the importance of glucose transporters in cancer cell motility [39].

Furthermore, our study identifies YAP1 as a transcriptional activator of SLC2A1. Previous research has established the pivotal role of YAP1 in cancer cell proliferation and survival through various metabolic pathways [40]. Jang et al. demonstrated that YAP1 can enhance the glycolytic phenotype by regulating glucose transporter expression [41]. More relevantly, Peng and colleagues have identified that YAP1 increases SLC2A1 expression in liver tumor to enhance glycolytic activity [19]. Our findings extend this knowledge by showing that YAP1 directly binds to the SLC2A1 promoter, significantly increasing its expression. This is particularly significant, as YAP1 is known to promote tumor growth in various cancers, including hepatocellular carcinoma [42] and breast cancer [43], by activating metabolic pathways essential for cancer cell survival. Additionally, studies in non-small cell lung cancer have shown that YAP1 overactivation contributes to metabolic reprogramming and resistance to therapies by upregulating glucose transporter expression [44].

Furthermore, the interaction between SLC2A1 and Wnt signaling underscores the interconnectedness of these regulatory mechanisms. Wnt signaling is frequently dysregulated in CRC, driving tumorigenesis through β-catenin-mediated transcription [45]. In the Wnt/β-catenin signaling pathway, dysregulated activity of the transcription factor β-catenin, a key component of the pathway, is implicated in the early stages of carcinogenesis [46]. Under normal conditions, when Wnt signaling is inactive, β-catenin is phosphorylated by GSK3β and casein kinase 1α, which leads to its ubiquitination and subsequent degradation via the proteasome [47, 48]. In this study, we observed that YAP1 silencing in CRC cells led to an increase in GSK3β levels, leading to reduced nuclear translocation of β-catenin and decreased levels of Wnt target genes. These effects were negated by the additional SLC2A1 silencing. This evidence indicates that the YAP1-SLC2A1 axis activates the Wnt/β-catenin pathway, at least partly, by reducing GSK3β levels and preventing β-catenin degradation.

Previous studies have shown that the glycolysis rate-limiting enzyme PFKP can promote the nuclear translocation of β-catenin [49]. Similarly, in oral cancer, knockout of Gherin inhibits GLUT1 and suppresses the nuclear translocation of β-catenin [50]. Interestingly, existing evidence has also indicated that the Wnt pathway activation is associated with increased glycolytic activity in cancer cells [51]. In pancreatic cancer, Wnt pathway components regulate GLUT1 expression, influencing tumor cell proliferation [52]. In ovarian cancer, Wnt pathway activation upregulates GLUT1, enhancing glucose uptake and supporting tumor cell metabolism [53]. Similarly, in head and neck squamous cell carcinoma, Wnt signaling has been implicated in regulating GLUT1 expression, further linking these pathways to tumor aggressiveness [54]. Our study extends these findings by connecting YAP1 to both SLC2A1 and Wnt signaling in CRC, highlighting the potential for targeted therapies that disrupt these pathways. Given these findings, it is plausible that a positive feedback loop exists between SLC2A1 and the Wnt/β-catenin pathway. However, due to time and financial constraints, we were unable to fully investigate whether this loop specifically operates within the context of CRC, which represents a limitation of this study.

Other limitations include the fact that, while our study provides evidence that the YAP1-SLC2A1-Wnt axis drives the malignant phenotype of CRC cells, the complexities of the tumor microenvironment may not be fully captured. Future investigations, particularly those using in vivo models, will be crucial to validate our findings and explore the therapeutic potential of targeting SLC2A1 and its regulatory networks in CRC. Additionally, although bioinformatics analysis suggested a link between SLC2A1 expression and poor patient outcomes, the lack of clinical samples may limit the translational relevance of our findings. We aim to address these gaps in future research.

Conclusion

In summary, our study highlights the pivotal role of SLC2A1 in CRC, emphasizing its regulation by YAP1 and its involvement in Wnt signaling. Targeting the YAP1-SLC2A1-Wnt axis may offer a promising therapeutic strategy for CRC. By addressing the metabolic dysregulation central to CRC progression, our findings contribute to the growing body of evidence supporting innovative treatments aimed at improving patient outcomes. Future research should focus on exploring the therapeutic implications of modulating SLC2A1 expression and its downstream effects on tumor growth and metastasis, paving the way for more effective interventions in CRC treatment.

Methods and material

Bioinformatics analysis

The GSE14297, GSE18462, and GSE40367 datasets were retrieved from the GEO database. Data processing was performed using R programming with the Limma package. A threshold of|Log Fold Change (FC) > 1.0| and an adjusted p-value < 0.05 were applied to identify differentially expressed genes (DEGs). Venn diagrams were created using the VennDiagram package in R to visualize the overlap of DEGs across the datasets. Additionally, SLC2A1 expression levels in CRC tissues were analyzed using the TCGA-COAD dataset, with the GTEx database providing baseline normal expression levels for comparison.

Cell culture

CRC cell lines Caco-2, HCT116, and SW480 (ATCC, USA) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and maintained at 37 °C in a humidified incubator with 5% CO₂. The immortalized normal colonic epithelial cell line FHC was cultured under the same conditions.

Cell transfection treatment

For SLC2A1 and YAP1 knockdown, two specific short hairpin RNAs (shRNAs) targeting each gene were designed (http://www.sabiosciences.com). The shRNA constructs were inserted into the pLKO.1 vector and transfected into Caco-2 and SW480 cells using Lipofectamine 2000 (Invitrogen). For overexpression studies, full-length SLC2A1 cDNA was cloned into the pcDNA3.1 vector. The resulting constructs were transfected into CRC cell lines. The gene silencing or overexpression in cells was confirmed by quantitative polymerase chain reaction (qPCR) and western blot (WB) analysis. For artificial activation of the Wnt signaling, CRC cells with SLC2A1 knockdown were seeded in 6-well plates and cultured overnight. Cells were then treated with SKL2001 (20 µM) or DMSO (vehicle control) for 24 h.

RNA reverse transcription and qPCR analysis

Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen). RNA concentration was measured with a NanoDrop spectrophotometer, and cDNA synthesis was performed using the SuperScript IV VILO Master Mix (Thermo Fisher). qPCR was carried out using SYBR Green Master Mix (Applied Biosystems) on a QuantStudio 7 Flex Real-Time PCR System. Gene expression was normalized to GAPDH, and relative expression levels were calculated using the 2−ΔΔCT method.

WB analysis

Cells were lysed in RIPA buffer (Roche), and protein concentrations were determined using the BCA kit (Thermo Fisher). Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes (Millipore). Membranes were blocked with 5% BSA for 1 h, then incubated overnight at 4 °C with primary antibodies against SLC2A1, YAP1, CTNNB1, c-Myc, c-JUN, and c-Met (Cell Signaling Technology). After washing, membranes were probed with HRP-conjugated secondary antibodies and detected using an ECL system (Thermo Fisher).

Luciferase reporter assays

The SLC2A1 promoter region (− 2000 to + 200 bp relative to the transcription start site) was cloned into the pGL3 vector. HEK293T cells were co-transfected with the luciferase reporter construct and a YAP1 overexpression vector. After 48 h of incubation, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). Data were normalized to Renilla luciferase activity, and results are presented as relative luciferase units.

Chromatin immunoprecipitation (ChIP)-qPCR

ChIP assays were performed to assess the binding affinity of YAP1 to the SLC2A1 promoter. Cells were cross-linked for 10 min with 1% formaldehyde, then quenched with 0.125 M glycine. Chromatin was sheared using a Bioruptor (Diagenode) to obtain fragments of approximately 200–500 bp. Immunoprecipitation was carried out with anti-YAP1 antibodies (Cell Signaling Technology) and protein A/G beads (Santa Cruz Biotechnology). The bound chromatin was eluted and analyzed by qPCR to quantify the enrichment of the SLC2A1 promoter using specific primers targeting the regions of interest.

Cell viability and proliferation assays

Caco-2 and SW480 cells were seeded in 96-well plates at a density of 1 × 10^4 cells per well and transfected with shRNAs or overexpression vectors. After 48 h, CCK-8 reagent was added, and absorbance was measured at 450 nm. For proliferation assays, EdU staining was performed using the Click-iT EdU Imaging Kit (Thermo Fisher). Cells were incubated with EdU for 2 h, fixed, and then treated with the EdU reaction cocktail. Staining was visualized under a fluorescence microscope, and the number of EdU-positive cells was quantified.

Apoptosis assays

Apoptosis was assessed using Annexin V/PI staining (BD Biosciences). Cells were resuspended in binding buffer, followed by the addition of Annexin V-FITC and PI. After 15 min of incubation in the dark, flow cytometric analysis was performed using a BD Accuri C6 flow cytometer. Data were analyzed using FlowJo software.

Migration and invasion assays

For migration assays, 1 × 105 cells were seeded in the apical chamber of a Transwell insert (8 μm pore size, Corning) without Matrigel. For invasion assays, the upper chamber was pre-coated with Matrigel (BD Biosciences). After 24 h, cells that migrated to the basolateral chamber were fixed with 4% paraformaldehyde, stained with crystal violet, and counted under a microscope.

Immunofluorescence staining

To evaluate CTNNB1 nuclear translocation, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% bovine serum albumin (BSA). Cells were then incubated with anti-CTNNB1 antibodies (Cell Signaling Technology) overnight at 4 °C, followed by incubation with Alexa Fluor-conjugated secondary antibodies (Invitrogen). Images were captured, and the percentage of CTNNB1 localized in the nucleus was quantified using ImageJ software.

TOP/FOP luciferase reporter assays

To assess the effect of YAP1 knockdown and SLC2A1 overexpression on Wnt signaling activity, we performed TOP/FOP luciferase reporter assays. Cells were seeded in 24-well plates and co-transfected with either TOP-Flash (Wnt-responsive) or FOP-Flash (mutant control) luciferase reporter plasmids along with Renilla luciferase (internal control). After 24 h, cells were further transfected with shYAP1, shCtrl, SLC2A1-OE, or vector control. After 48 h of transfection, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s protocol. Firefly luciferase activity was normalized to Renilla luciferase activity, and the relative TOP/FOP ratio was calculated.

Statistical analysis

All experiments were performed in triplicate, and data are presented as mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism software. Comparisons between groups were made using One-Way or Two-Way ANOVA, with p < 0.01 considered statistically significant.

Data availability

No datasets were generated or analysed during the current study.

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Authors and Affiliations

  1. Zhongda Hospital of Southeast University, No 87 Dingjiaqiao, Nanjing, 210009, Jiangsu, PR China

    Kunpeng Li

  2. Department of General Surgery, Zhongda Hospital, Southeast University, Nanjing, 210009, Jiangsu, PR China

    Ya-Jie Dai, Haifeng Zhang & Zhigang Zhang

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Kunpeng Li: Conceptualization, Methodology, Data Curation, Writing– Original Draft Preparation. Ya-Jie Dai: Investigation, Formal Analysis, Visualization, Writing– Review and Editing. Haifeng Zhang: Validation, Resources, Supervision, Writing– Review and Editing. Zhigang Zhang: Project Administration, Funding Acquisition, Supervision, Writing– Final Review.

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Li, K., Dai, YJ., Zhang, H. et al. YAP1 activates SLC2A1 transcription and augments the malignant behavior of colorectal cancer cells by activating the Wnt/β-catenin signaling pathway. Cell Div 20, 8 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13008-025-00148-y

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Keywords

Cell Division

ISSN: 1747-1028

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