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. 2024 Jan 26;115(4):1154–1169. doi: 10.1111/cas.16082

Itraconazole inhibits tumor growth via CEBPB‐mediated glycolysis in colorectal cancer

Yong Zhang 1,2,3,4, Lu Li 2,3,4, Feifei Chu 2,3,4, Huili Wu 2,3,4, Xingguo Xiao 2,3,4, Jianping Ye 1,✉, Kunkun Li 2,3,4,✉
PMCID: PMC11007002  PMID: 38278779

Abstract

Advanced colorectal cancer (CRC) is characterized by a high recurrence and metastasis rate, which is the primary cause of patient mortality. Unfortunately, effective anti‐cancer drugs for CRC are still lacking in clinical practice. We screened FDA‐approved drugs by utilizing targeted organoid sequencing data and found that the antifungal drug itraconazole had a potential therapeutic effect on CRC tumors. However, the effect and mechanism of itraconazole on CRC tumors have not been investigated. A cell line‐derived xenograft model in tumor‐bearing mice was established and single‐cell RNA sequencing was performed on tumor samples from four mice with or without itraconazole treatment. The proportion of cell populations and gene expression profiles was significantly different between the two groups. We found that itraconazole could inhibit tumor growth and glycolysis. We revealed that CEBPB was a new target for itraconazole, and that silencing CEBPB could repress CRC glycolysis and tumor growth by inhibiting ENO1 expression. Clinical analysis showed that CEBPB expression was obviously elevated in CRC patients, and was associated with poor survival. In summary, itraconazole treatment remodeled cell composition and gene expression profiles. Itraconazole inhibited cell glycolysis and tumor growth via the CEBPB–ENO1 axis. In this study, we illustrate a new energy metabolism mechanism for itraconazole on tumor growth in CRC that will provide a theoretical basis for CRC targeting/combination therapy.

Keywords: CEBPB, colorectal cancer, glycolysis, itraconazole, single‐cell RNA sequencing

Itraconazole treatment remodeled cell composition and gene expression profiles. Itraconazole inhibited CRC tumor growth via cell glycolysis and the CEBPB–ENO1 axis. CEBPB expression was elevated in CRC and was associated with poor survival.

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1. INTRODUCTION

CRC is one of the most common tumor types in the digestive system with high morbidity and mortality in recent years. 1 At present, radical surgery combined with chemoradiotherapy can benefit CRC patients in the short term, but most patients still relapse, with metastasis and chemotherapy resistance within several years after treatment, resulting in a poor prognosis for patients. 2 Although large fruitful achievements have been made in pathogenesis and clinical transformation research in recent years, CRC currently still lacks an effective therapeutic schedule in the clinic, 3 so there is an urgent need to explore novel anti‐cancer drugs and therapeutic biomarkers to serve clinical needs. In this study, by analyzing organoid‐targeted sequencing data under the conditions of drug interference, we screened the potential antitumor drug itraconazole from FDA‐approved drugs widely used in the clinic, and assessed its impact on the viability of CRC tumors.

Itraconazole is widely used clinically as an antifungal drug. It can inhibit lanosterol 14α‐demethylase, a key enzyme in the process of ergosterol synthesis, and results in the depletion of ergosterol in the fungal cell membrane and the accumulation of methyl sterol, thus inhibiting fungal growth by destroying the integrity of the membrane. 4 Recent studies have found that itraconazole has an inhibitory effect on tumor growth, invasion and metastasis in multiple cancer types, which has been confirmed in non–small‐cell lung cancer studies and other clinical trials. 5 Itraconazole treatment is an independent protective factor for 5‐year survival in CRC patients. 6 Currently, the antitumor mechanism of itraconazole mainly includes the following aspects: Wnt/Hedgehog‐mediated cell cycle arrest, Bcl2/Cyt‐c‐mediated apoptosis, VEGFR2‐mediated anti‐angiogenesis and PI3K–AKT–mTOR‐mediated autophagy and ABCC1‐mediated drug resistance inhibition, 7 as well as killing dormant cancer cells. 8 However, the effect and mechanism of itraconazole on CRC tumors have not been reported at the cellular level. Therefore, we intended to investigate the effect and mechanism of CRC tumor growth in a mouse model combined with single‐cell sequencing technology.

Solid tumors adapt to the extreme tumor microenvironment by reprogramming energy metabolism, especially the abnormal activation of the aerobic glycolysis pathway. 9 A body of evidence has shown that, due to the rapid growth of tumor cells, tumors exist in a hypoxic environment, which directs tumor cells to use aerobic glycolysis to obtain energy and produce large amounts of the metabolic byproduct lactic acid in the cytoplasm; this is known as the “Warburg effect.” 10 The acidic environment of the extracellular matrix promotes further proliferation and invasion of tumor cells and avoids apoptosis, while protecting tumor cells from attack by the immune system. It is the activation of aerobic glycolysis that hinders the effectiveness of current treatments for CRC, so blocking aerobic glycolysis is currently considered a promising cancer treatment strategy. 11 Enolase 1 (ENO 1) is an enolase isoenzyme that promotes the formation of phosphoenolpyruvate through the catalytic action of 2‐phosphoglycerate and the production of ATP(4) during glycolysis. 12 ENO1 has been demonstrated to be highly expressed in multiple tumors and is often associated with a poor prognosis for cancer patients. ENO1 overexpression could promote tumorigenesis, proliferation, metastasis, and tumor stemness. 13 Therefore, ENO1 can be used as a potential therapeutic target to control the energy metabolism of tumors. 14

In our study, we found that itraconazole could repress the expression of a new transcription factor (TF) Enhancer Binding Protein Beta (CEBPB), thus inhibiting glycolysis and tumor growth by controlling ENO1 expression. A series of evidence unveiled that the CEBP family was an important TF in tumors, 15 including CRC. 16 However, the role of CEBPB in CRC on aerobic glycolysis remains unclear, especially the target gene of itraconazole. Hence, this study aims to investigate the influence of itraconazole targeting CEBPB on CRC, and whether CEBPB protein targeting ENO1 expression affects tumor growth through aerobic glycolysis.

2. MATERIALS AND METHODS

2.1. Public data sources

RNA‐seq data from GSE114012 (two CRC cell lines SW948 and HT55 with itraconazole treatment) were used to detect potential target genes for itraconazole. The RNA and protein expression data, as well as the clinical data, of 622 and 197 CRC patients in the cancer genome atlas (TCGA) 17 and clinical proteomic tumor analysis consortium (CPTAC) were used. 18

2.2. Cell culture and transfection

The human CRC cell line HCT116 was obtained from Procell (Wuhan, China) and the National Infrastructure of Cell Line Resource (NICR), respectively. HCT116 cells were cultured in McCoy 5A (Procell, China) and IMDM (Procell, China) medium containing 10% FBS, respectively. Cells were maintained in a wet incubator with 5% CO2 at 37°C. The recombinant lentivirus used for CEBPB knockdown (sh‐CEBPB#1, #2) and the corresponding control lentivirus (sh‐NC) were purchased from GeneChem (Shanghai, China). The reagent HiTransG A (GeneChem, China) was utilized to transfect lentiviruses into CRC cells.

2.3. Single‐cell RNA sequencing (scRNA‐seq) and bioinformatics analysis

The tumor samples with or without treatment with itraconazole from the HCT116‐derived CDX mouse model were utilized for single‐cell RNA sequencing and bioinformatics analysis (2 vs. 2). The dually edited Mel‐RM (Mel‐RM.DE) cells were serum starved for 96 h, and the endometrial quiescent cells were sorted, washed twice and resuspended in cold PBS (calcium and magnesium free) containing 0.04% FBS. Cell number and viability were determined using a hemocytometer and trypan blue staining, and 1 × 105 cells were subjected to 10× Genomics sequencing according to the manufacturer's protocol by Shanghai OE Biotech Co., Ltd. (Shanghai, China). Briefly, viable endometrial cells isolated from dually edited Mel‐RM cells after serum starvation were analyzed using the 10× Genomics Chromium Droplet platform with unique transcript counting through barcoding with unique molecular identifiers (UMIs). Cell Ranger 3.1.0 and Seurat 3.1.1 were used to analyze the sequencing results.

Considering that these samples were from the HCT116‐derived CDX mouse model, we mapped the sequenced reads to human and mouse reference genomes. After quality filtering to remove cells expressing high mitochondrial gene signatures and excluding doublets, 45,399 cells were retained for further analysis. Upon gene expression normalization for read depth, cells were subjected to uniform manifold approximation and projection (UMAP) or t‐distributed stochastic neighbor embedding (tSNE) and several unsupervised cell clusters were obtained and visualized using the Loupe browser. The cluster‐specific markers were identified by detecting the differentially expressed genes between the given cluster and the other clusters.

To identify genes differentiating the itraconazole‐treated and DMSO groups, differentially expressed genes were first identified using the R package Seurat (Fold Change >1.5 or <1/1.5 and a false discovery rate (FDR) < 0.05). Gene Ontology (GO) terms and KEGG pathways were enriched using function enrichment analysis with a cutoff of p < 0.05. In addition, in order to identify GO terms, KEGG pathways and cancer hallmarks alternatively, all genes sorted to differentially expressed degrees (Fold Change) were used for Gene Set Enrichment Analysis 19 (GSEA). Function enrichment analysis and GSEA were implemented using the MSigDB 20 database and R package clusterProfiler. 21

2.4. qRT‐PCR and western blot

Our in‐house CRC cohort included 37 pairs of fresh CRC tumor and adjacent normal tissue specimens without radiotherapy or chemotherapy, which were immediately stored in liquid nitrogen after surgery (Table S1). All specimens were collected from Zhengzhou Central Hospital Affiliated to Zhengzhou University between 2019 and 2020 and this study was approved by the Zhengzhou Central Hospital Affiliated to Zhengzhou University. All patients had undergone rigorous screening and underwent informed consent.

qRT‐PCR was employed to detect the RNA levels of CEBPB, ENO1 and β‐Actin. Table S2 lists the involved primer sequences. Table S3 shows the relative expression level of CEBPB and ENO1 mRNA in 37 pairs of CRC tumors and matched to adjacent normal samples. Western blotting was used to detect the protein levels of corresponding genes in 10 pairs of tumors and the matched adjacent normal samples from our center. Primary antibodies used in this study included anti‐β‐Actin (Proteintech, HRP6008), anti‐β‐Tubulin (Abcam, ab179511), anti‐CEBPB (Abcam, ab32358), and anti‐ENO1(CST, 3810T). Table S4 shows the relative expression levels of CEBPB and ENO1 proteins in 10 pairs of CRC tumors and the matched adjacent normal samples (see more in Appendix S1).

2.5. The detection indexes of glycolysis

This study determined glucose and the activity of the glycolysis pathway by quantitatively measuring the levels of different products (including pyruvate, and lactic acid) produced during glycolysis. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse Biosciences XFe96 analyzer (Agilent). Cells were cultured in Petri dishes for 24 h, and then cultured in XF medium at 37°C for 2 h. OCR was measured complying with the XF Cell Mito Stress Test Profile instructions. Oligomycin, FCCP and antimycin A and rotenone were added in sequence, and then the OCR value was determined. ECAR was measured by complying with the XF Glycolysis Stress Test Profile. Glucose, oligomycin and 2‐deoxyglucose (2‐DG) were added sequentially to each well until the concentration reached a specified level. The ECAR value represents glycolysis capability, while the OCR value represents the oxygen consumption rate.

2.6. The establishment and disposition of the CDX mouse model

To evaluate the effect of itraconazole on CRC tumors, HCT116 cells (5 × 106) were cultured and inoculated subcutaneously into 12 nude mice (6 weeks of age, 16–18 g) until the subcutaneous tumor grew to about 100 mm3. The mice were divided randomly into two groups with six samples in each group. There was no statistical significance in tumor size between the two groups. Subsequently, itraconazole (50 mg/kg) dissolved in DMSO solution (5% DMSO + 40% PEG300 + 10% Tween 80 + 45% ddH2O) or DMSO solution was injected into the abdomen six consecutive times, once every other day, and the tumor size was measured at two intervals. The mice were sacrificed 3 days after the last injection, and tumor size and weight were recorded.

2.7. Chromatin immunoprecipitation (ChIP) assay

CRC cells were cultured in a 100‐mm Petri dishes to 80% confluency. ChIP assay was undertaken following the ChIP kit protocol (P2078, Beyotime). Cell lysates were cultured with IgG as a control. PCR was applied to amplify the ENO1 promoter region (located in chr1:8878,600–8878,880) that contained the putative CEBPB binding site. Antibodies used in the ChIP assay were anti‐CEBPB (ab32358) and anti‐IgG (ab109489). Table S5 shows the primer sequences used in the ChIP assay.

3. RESULTS

3.1. The screening of itraconazole in CRC tumors

We screened antitumor drugs in CRC organoids and cell lines using large‐scale drug interference sequencing and cell viability data, and ultimately determined itraconazole (Figure 1A). Especially, expression changes for 206 genes reflecting the phenotype and function of intestinal epithelial differentiation were detected by organoid‐targeted sequencing for CRC tumors treated by 320 FDA‐approved drugs. 22 In total, 109 candidate drugs with significant expression changes of at least five genes were identified through differential expression analysis (Figure 1B). Then seven drugs showed an increased proportion of enterocyte epithelial cells, and a decreased proportion of intestinal stem cells (ISCs) after treatment (Figure 1C), indicating that they may exert an antitumor effect based on differentiation therapy. Subsequently, three cell lines representing different molecular subtypes of CRC, namely DLD1, LS174T, and HCT116, were selected to evaluate the antitumor effect of drugs (Figure 1D), and drugs with significant inhibitory effects on cell viability were determined. Finally, the LINCS‐L1000 and TCGA/GEO datasets were used to identify potential drugs in CRC cell lines based on the expression reversal method, and the top 20 drugs were selected according to the rank score calculated in multiple datasets. It was found that only itraconazole could accord with these four conditions simultaneously (Figure 1E). Thus we focused our attention on itraconazole.

FIGURE 1.

FIGURE 1

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The screening of itraconazole from CRC organoid sequencing and cell viability assay. (A) General process for screening itraconazole. (B) In total, 109 drugs were screened from organoid‐targeted sequencing data. The top 20 drugs were closely related to the expression changes of at least 10 genes related to intestinal epithelial differentiation phenotype and function. (C) Compared with blank control treatment, the proportion changes of eight cell types in intestinal organoids after drug treatment, among which DC1 and DC2 are two known drugs that clearly induce a decrease in intestinal stem cell characteristics and an increase in epithelial cell characteristics. (D) FACS measures the mortality rate of three CRC cell lines after drug treatment. Annexin and DAPI are used to measure the multiples of cell mortality in the drug treatment compared with the blank control treatment. (E) Transcriptomic data from LINCS‐L1000, TCGA and GEO datasets are used to identify potential therapeutic agents in CRC cell lines based on the expression reversal method.

3.2. The antitumor effect of itraconazole in CRC

To evaluate the effect of itraconazole on CRC tumors, an HCT116‐derived CDX mouse model was established and treated with itraconazole or DMSO solution (control) (Figure 2A). Compared with the control group, the tumor size and weight in the itraconazole‐treated group were significantly reduced (Figure 2B–E), indicating its inhibitory effect on the growth of CRC tumors. Subsequently, single‐cell RNA sequencing (scRNA‐seq) was performed on tumor xenografts. Functional enrichment analysis showed that classical tumor phenotypes and functions (DNA replication, cell adhesion and apoptosis) were affected by itraconazole treatment (Figure 2F–H). These results further suggested that itraconazole could inhibit CRC tumor growth by influencing cell proliferation and apoptosis.

FIGURE 2.

FIGURE 2

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Experimental validation of the itraconazole effect on CRC tumors and scRNA‐seq analysis. (A) Workflow of animal experiments and processing for scRNA‐seq. (B, C) Tumor size of mice treated with itraconazole or DMSO. (D) Tumor weight of itraconazole‐treated and control groups after isolation. (E) Tumor volume of itraconazole‐treated and control groups at different time points. (F–H) GSEA plot of enriched GO terms, KEGG pathways and cancer hallmarks.

3.3. Itraconazole remodeled the cell composition and expression profiles

To investigate the tumor heterogeneity in the conditions of itraconazole treatment at the cell level, scRNA‐seq was conducted on tumor xenografts from four mice treated with itraconazole or DMSO. After quality control, 31,165 mouse‐derived cells were obtained. After principal component analysis (PCA) and dimension reduction clustering, 15 distinct cell clusters were visualized by UMAP (Figure 3A). The cell annotation analysis showed that these cell clusters corresponded to eight cell types, including epithelial cells (n = 22,016, 70.6%), myeloid cells (n = 4219, 13.5%), cancer‐associated fibroblasts (CAFs) (n = 3290, 10.6%), endothelial cells (n = 1000, 3.2%), myofibroblasts (n = 313, 1%), Neutrophils (n = 103, 0.3%), NK cells (n = 99, 0.3%) and tumor stem cells (CSCs) (n = 125, 0.4%) (Figure 3B–E; Figure S1). Compared with the control group, there was an obvious increase in the proportion of epithelial cells and a decrease in the proportion of CAFs, myofibroblasts and myeloid cells in the itraconazole‐treated group (Figure 3F).

FIGURE 3.

FIGURE 3

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Itraconazole remodeled the cell composition and expression profiles. (A) UMAP plot of 15 clusters after dimension clustering based on scRNA‐seq data. (B) Annotated cell types corresponding to cell clusters. (C) Proportion of each cell type. (D, E) Expression of myeloid cell‐specific and endothelial cell‐specific marker genes. (F) Difference in the proportion of all cell types between the two groups. (G) Heatmap of metabolism signaling activity in the two groups.

Reprogramming of core cellular metabolic pathways by cancer cells provides energy, anaplerotic precursors and reduces equivalents required to support CRC tumor growth. 23 Thus we further analyzed the activity of metabolic pathways using scMetabolism. 24 Compared with the control group, there was a significant decrease in the activity of glycolysis/gluconeogenesis, pentose phosphate pathway (PPP) and pyruvate metabolism, while there was a significant increase in the activity of taurine, phosphonate, nicotinamide and caffeine metabolism in the itraconazole‐treated group (Figure 3G). Large‐scale studies have shown that glycolysis and PPPs as well as pyruvate metabolism are significantly enhanced in tumors, thus providing energy and the substances required for biosynthesis for tumor cell proliferation and metastasis, 25 , 26 and providing new evidence for treating CRC tumors by inhibiting these pathways.

3.4. Itraconazole repressed CRC tumor growth by inhibiting glycolysis

To evaluate the new therapeutic mechanism of itraconazole on CRC tumors, differential expression analysis and functional enrichment analysis were conducted based on the scRNA‐seq data. We found that the differentially expressed genes were significantly enriched in glycolysis and gluconeogenesis using GO terms and KEGG pathways (Figure 4A,B). Especially, the expression of several promoting‐glycolysis enzymes (ENO1, LDHA, PGK1, PKM and GAPDH) was significantly decreased after itraconazole treatment (Figure 4C,D). Although the effect of itraconazole on the CRC cell cycle and apoptosis is well established, its effect on glucose metabolism has not yet been investigated. Thus we speculated that itraconazole may inhibit tumor growth by affecting glycolysis and further detected the glycolysis‐relevant indicators in tumor tissues.

FIGURE 4.

FIGURE 4

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Itraconazole inhibited the tumor growth and glycolysis in CRC. (A, B) Function enrichment analysis based on GO terms and KEGG pathways. (C) Heatmap of differentially expressed genes in itraconazole‐treated and control groups. (D) Expression of ENO1, LDHA, PGK1, PKM and GAPDH in the two groups. (E–G) Content of glucose uptake, pyruvate and lactic acid in itraconazole‐treated and control groups. (H, I) Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were detected by XFe96 Seahorse in the itraconazole‐treated and control groups. (J, K) Basal glycolysis, glycolysis capacity, maximum oxygen consumption and respiratory potential from ECAR and OCR in the itraconazole‐treated and control groups.

Compared with the control group, we observed that the levels of glucose uptake, pyruvate and lactic acid in the itraconazole‐treated group were obviously decreased (Figure 4E–G). Seahorse energy analysis further showed that itraconazole treatment could significantly reduce ECAR and OCR (Figure 4H,I), as well as basal glycolysis, glycolysis capacity, maximum oxygen consumption and respiratory potential (Figure 4J,K), which suggested that aerobic glycolysis was inhibited. These results suggested that itraconazole could repress CRC tumor growth by inhibiting glycolysis.

3.5. CEBPB was a new target of itraconazole

In our scRNA‐seq data, 61 potential target genes of itraconazole (26 upregulated and 35 downregulated) that were significantly differentially expressed were identified (Figure 5A). Then we also identified 136 differentially expressed genes (52 upregulated and 84 downregulated) in HT55 and SW948 cell lines treated with itraconazole from the GSE114012 dataset. By intersecting the two differential expression gene lists mentioned above, we found that CEBPB was the only target gene that was consistently downregulated after itraconazole treatment (Figure 5B). Compared with the control group, the proportion of expression cells and the expression abundance of CEBPB in the itraconazole‐treated group were significantly decreased (Figure 5C,D), and CEBPB was expressed extensively in multiple cell types, especially myeloid cells (including monocytes, macrophages and dendritic cells) (Figure 5E). Through SCENIC analysis, 27 we found that CEBPB was the main TF that exerted a regulatory role in myeloid cells and neutrophils (Figure 5F). Meanwhile, CEBPB was the potential TF that mediated the transcriptional network in control tumors, whereas it disappeared in itraconazole‐treated tumors (Figure 5G). These results suggested that CEBPB was the potential target of itraconazole in CRC.

FIGURE 5.

FIGURE 5

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CEBPB was identified as the downstream target gene of itraconazole. (A) Volcano plot of differentially expressed genes between itraconazole‐treated and control groups. (B) mRNA expression changes in CEBPB were detected in itraconazole‐treated HT55 and SW948 cell lines from the GSE114012 dataset. (C) Proportion of expression cells for CEBPB between the two groups. (D) Expression abundance of CEBPB between two groups. (E) Expression distribution of CEBPB in different cell types from the two groups. (F) Heatmap of TF activity in different cell types. (G) Regulon specificity score (RSS) ranking plot of the top five TFs in the two groups. The regulator with a higher RSS may be related to the specificity of this group. (H, I) mRNA and protein expression of CEBPB treated with itraconazole was detected. (J) Representative western bolt of CEBPB in the above two groups.

Next, qRT‐PCR and western blot were used to detect the mRNA and protein expression levels of CEBPB in the control and itraconazole‐treated groups (HCT116 cells and mice tumor tissue). We found that the mRNA and protein expression levels of CEBPB were significantly decreased in the itraconazole‐treated group (Figure 5H–J), which confirmed that CEBPB was a new target for itraconazole to exert a therapeutic effect.

3.6. CEBPB was highly expressed in CRC and associated with clinical significance

We first studied the mRNA expression and clinical significance of CEBPB in CRC patients from TCGA project, and found that, compared with adjacent normal tissues, CEBPB expression was significantly higher in tumors (Figure 6A). The higher the expression of CEBPB, the more malignant the tumor stage for the CRC patient (Figure 6B), which was in agreement with the prognostic significance of the CEBPB protein in CRC. 28 Moreover, high expression of CEBPB was significantly correlated with shorter overall survival (OS) time in CRC patients (Figure 6C). Furthermore, in the CPTAC project, the protein expression of CEBPB in tumors was also higher than in normal samples (Figure 6D). Also the later the tumor stage, the higher the expressed CEBPB protein level (Figure 6E). Immunohistochemistry (IHC) results from The Human Protein Atlas (HPA) databases also confirmed the high expression of CEBPB protein in CRC patients (Figure 6F). CEBPB was mainly expressed in the nucleus, suggesting the possibility of regulating the expression of target genes as transcription factors.

FIGURE 6.

FIGURE 6

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CEBPB showed high expression in CRC tumors, and was associated with clinical significance. (A) mRNA expression of CEBPB from CRC tumors and adjacent normal samples in TCGA. (B) Association between CEBPB mRNA expression and tumor TNM stage in TCGA. (C) Relationship between CEBPB mRNA expression and patient overall survival. (D) Protein expression of CEBPB from CRC tumors and adjacent normal samples in CPTAC (E) Association between CEBPB protein expression and tumor TNM stage in CPTAC. (F) Representative image of CEBPB protein expression detected by IHC from the HPA database in CRC tumors and normal intestinal epithelia. (G) CEBPB protein expression level in NCM460 and 7 CRC cell lines. (H) Western blot of CEBPB in above cell lines. (I, J) CEBPB mRNA and protein expression level in CRC tumors and matched normal samples from the inner dataset. (K) Western blot of CEBPB in tumors and matched normal samples.

Western blotting was used to detect CEBPB expression protein levels in NCM460 and seven CRC cell lines, and it was found that CEBPB expression was significantly increased in CRC (Figure 6G,H). We then measured mRNA and protein levels in surgical samples (tumor and matched adjacent normal tissue) from 37 CRC patients and 10 CRC patients using qRT‐PCR and western blotting, respectively. Compared with matched adjacent normal tissues, mRNA and protein expression of CEBPB were significantly upregulated in tumor tissues (Figure 6I–K). These results suggested that CEBPB was an oncogene in CRC tumor progression.

3.7. CEBPB knockdown repressed the cell proliferation and glycolysis

We selected HCT116 cells for the conducted subsequent experiments, as they showed the most obvious increase in CEBPB expression compared with NCM460, and conducted knockdown experiments with two siRNAs (Figure 6G). To explore the impact of aberrant expression of CEBPB on tumor proliferation, si‐CEBPB cell lines were established. First, qRT‐PCR results showed that CEBPB expression was notably reduced in cells transfected with si‐CEBPB (Figure 7A), indicating the eligible cell transfection efficiency. Then the effect of CEBPB on the cell proliferation ability in vitro was detected by CCK‐8, EdU and colony formation assays (see more in Appendix S1). The results showed that the knockdown of CEBPB significantly inhibited the tumor cell viability in vitro (Figure 7B–D), which had been demonstrated in previous animal experiments, 29 indicating that CEBPB played an oncogene role in CRC. Compared with the control group, the contents of glucose, pyruvate and lactic acid in si‐CEBPB group were significantly decreased (Figure 7E–G). Furthermore, ECAR and OCR (Figure 7H,I), as well as the basal glycolysis, glycolysis capacity, maximum oxygen consumption and respiratory potential (Figure 7J,K) were significantly restrained when CEBPB was knocked down. These results suggested that silencing CEBPB could inhibit tumor proliferation and glycolysis in CRC.

FIGURE 7.

FIGURE 7

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Knockdown of CEBPB inhibited cell proliferation and glycolysis in vitro. (A) mRNA and protein expression of CEBPB after transfecting si‐CEBPB or control into HCT116 cell. (B) Cell viability was detected by CCK‐8 assay. (C, D) Cell proliferation ability detected by clonal formation and EdU assay. (E–G) Content of glucose, pyruvate and lactic acid in itraconazole‐treated and control groups. (H, I) ECAR and OCR change detected by Seahorse XFe96. (J, K) Basal glycolysis, glycolysis capacity, maximum oxygen consumption and respiratory potential were calculated based on ECAR and OCR.

3.8. CEBPB activated glycolysis by transcriptionally regulating ENO1

To investigate how CEBPB regulates the glycolysis process, we analyzed the potential regulation relationships in the hTFtarget database, and found that ENO1, LDHA, PGK1, PKM and GAPDH were positively regulated by transcriptional activator CEBPB. ChIPBase and JASPAR databases were adopted to determine the relationship between CEBPB and these potential target genes. ENO1 was the only glycolytic enzyme regulated by CEBPB in CRC cell lines, whose tumor‐promoting effect has been widely validated in CRC. 12 , 30 By detecting the effect of CEBPB knockdown on ENO1 expression, we found that CEBPB knockdown in HCT116 significantly decreased the expression of ENO1 at mRNA and protein levels (Figure 8A–C). Compared with the control group, the protein expression of ENO1 was obviously decreased in the itraconazole‐treated group (HCT116 cells and mice tumor tissue) (Figure 8D–F), suggesting that the glycolytic effect of CEBPB may be inhibited by itraconazole in CRC tumors. In our inner cohort, compared with adjacent normal tissues, ENO1 expression was significantly higher in tumors at mRNA and protein levels (Figure 8G–I), which was confirmed in TCGA and the CPTAC project (Figure 8J,K). Furthermore, to investigate the transcriptional regulation of CEBPB on its target gene ENO1, the ChIP‐seq data in HCT116 from the ENCODE project showed that the CEBPB protein had an obvious binding peak in the ENO1 DNA promoter region (Figure 8L). Finally, the ChIP‐PCR assay verified that CEBPB bound to the ENO1 promoter region in the tumor, while the binding intensity was evidently attenuated under itraconazole treatment conditions (Figure 8M). This result implied that CEBPB transcriptionally regulated ENO1, activated glycolysis and promoted CRC tumor initiation and growth.

FIGURE 8.

FIGURE 8

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CEBPB regulated glycolysis by affecting ENO1 expression. (A–C) Expression change of ENO1 at the RNA and protein level in itraconazole‐treated and control groups. (D–F) Expression change of ENO1 at the RNA and protein level in si‐NC and si‐CEBPB groups. (G, H) ENO1 expression at the mRNA and protein level in tumor and adjacent normal tissues from the inner dataset. (I) Western blot of ENO1 in tumors and matched normal samples. (J, K) ENO1 mRNA and protein expression in tumors and normal tissues from TCGA and CPTAC datasets. (L) Peak of ENO1 promoter region binding to CEBPB in HCT116 cells from the ENCODE project (GEO accession numbers: GSM5463600 and GSM5463601). (M) Intensity of CEBPB binding to the ENO1 promoter region in the itraconazole‐treated and control groups.

4. DISCUSSION

In this study, we revealed that CEBPB played a role as an oncogene in CRC. CEBPB promoted CRC aerobic glycolysis by regulating the expression of rate‐limiting enzyme ENO1; itraconazole could inhibit the pro‐tumor effect of CEBPB, thus repressing tumor growth. This study investigated the role of itraconazole on the biological behavior of CRC cells, as well as the mechanism of inhibition of CRC aerobic glycolysis through the CEBPB–ENO1 axis by sequencing and experiments. The study will help to reveal new mechanisms of CRC energy metabolism and new strategies for targeted therapy.

In our study, we found that itraconazole treatment could remodel gene expression and cellular composition, including immune‐related cells. The following aspects may account for the findings: (1) itraconazole regulates the lipid metabolism of macrophages and polarization of macrophages 31 , 32 ; (2) itraconazole exerts anti‐angiogenesis by stimulation of apoptosis in endothelial cells, repression the motility of endothelial cells or inhibiting CAF proliferation 33 , 34 , 35 ; (3) itraconazole interferes with the uptake of calcium by activated neutrophils by alteration of glycosylation or glycoprotein processing. 36 , 37 Thus, we speculate that itraconazole may remodel cell composition by directly or indirectly affecting the number or function of macrophages, endothelial cells and neutrophils.

Compared with the control group, there was a significant decrease in the activity of glycolysis/gluconeogenesis, PPP and pyruvate metabolism in the itraconazole‐treated group (Figure 3G). Large studies have shown that glycolysis and PPP as well as pyruvate metabolism are significantly enhanced in tumors, thus providing the energy and substances required for biosynthesis for tumor cell proliferation and metastasis, 25 , 26 and providing new evidence for treating CRC tumors by inhibiting these pathways. In our results, the activity of the PPP was inhibited by itraconazole treatment. In tumor cells, PPP is always activated and large amounts of glucose are consumed through the bypass. However, this pathway cannot produce the energy needed for cell growth, but can only be used for mass biosynthesis, producing large amounts of pentose (components of nucleotides) and reducing agent NAPDH (needed for fat synthesis) to satisfy the rapid and infinite growth of tumor cells. 38 Ribose‐5‐phosphate (R5P) in cells is an intermediate metabolite of PPP for de novo and remedial nucleotide synthesis during the cell cycle, and a large amount of R5P is required for abnormal proliferation of tumor cells. 39 Thus, the decreased activity of PPP after itraconazole treatment indicated the inhibition of tumor proliferation.

The treatment of CRC has become a difficult problem in the medical field due to its own characteristics of malignant proliferation, invasion and metastasis. In the process of tumor evolution, tumor cells adapt to the tumor microenvironment through an energy‐reprogramming metabolic process. It has been confirmed that abnormal activation of aerobic glycolysis pathway is a significant feature of energy metabolism in CRC. 40 The acidic environment of the extracellular matrix caused by lactic acid accumulation during aerobic glycolysis further promotes the proliferation and invasion of tumor cells and escapes normal cell apoptosis, while protecting tumor cells from the attack of the immune system. 41 Therefore, inhibition of aerobic glycolysis is currently generally considered a promising tumor treatment strategy. 42 Although there is an indirect clue accounting for the connection between itraconazole and the Warburg effect: itraconazole could inhibit glycolysis by targeting PfLDH and regulating its activity in falciparum. 32 However, direct studies on the effects of itraconazole on glycolysis in mammals are lacking. In our study, we found that itraconazole could repress CRC tumor growth by inhibiting aerobic glycolysis. Therefore, this is the first study to directly prove the effect of itraconazole on the Warburg effect, which provides new theoretical guidance for itraconazole targeting tumor energy metabolism.

CEBPB is previously implicated in the suppressive activity of myeloid‐derived suppressor cells (MDSC). 43 MDSC can suppress the tumor immune response, including inhibiting the functions of CD8+/CD4+ T cells, T cells, NK cells and B cells, and promoting the transformation of CD4+ T cells into Tregs (regulatory cells), which makes the tumor achieve immune escape, 44 thus promote tumor angiogenesis, tumor invasion and metastasis. The activated CEBPB promotes immunosuppression of MDSC by binding to and affecting the immunosuppressive enzymes (including Arg1, Nos2, Nox2 and Cox2). 45 CEBPB is also involved in M2 macrophage polarization. M2‐type macrophages exist in the microenvironment of most tumors, and play a pro‐tumor role in tumor growth, invasion and metastasis. 46 , 47 CEBPB together with its regulator CREB mediate the expression of M2 phenotype genes (such as Arg1 and Mrc1), inducing M2 macrophage polarization. 48 In our results, CEBPB was mainly expressed in myeloid cells (including monocytes, macrophages and dendritic cells). Its expression was significantly increased from normal to tumor, and was reversed after itraconazole treatment. Therefore, it is a potential antitumor strategy to weaken or even eliminate the immunosuppressive effect of MDSC by inhibiting the activity of CEBPB. Meanwhile, inhibition of CEBPB may lead to decreased polarization of M2 macrophages and exert antitumor activity.

Large studies have revealed that itraconazole in combination with other current agents made cancer patients benefit from oncotherapy in laboratory and clinical trials. 4 , 49 , 50 For example, combination chemotherapy with gemcitabine, nab‐paclitaxel, oxaliplatin and itraconazole (GnPO‐ITC) regimen showed promising efficacy with manageable toxicities for controlling disease progression and improving OS in pancreatic cancer patients. 51 Through inhibiting hedgehog signaling and angiogenesis, and inducing apoptosis and autophagy and reversing multidrug drug resistance by inhibiting p‐glycoprotein, 4 itraconazole can enhance the chemotherapeutic sensitivity of other drugs and alleviate their chemotherapy resistance. Currently, only one recent study revealed that the combination of itraconazole and anti‐PD‐1 antibody effectively suppressed tumor growth in head and neck cancer. 52 We expect to see more clinical trials of itraconazole combined with chemotherapy drugs and even immuno‐check inhibitors for eliminating the tumor, repression or remission of tumor progression.

In summary, our study demonstrated that itraconazole treatment could repress CRC tumor growth and glycolysis by regulating ENO1 expression. Combined with sequencing and experiments, we found that CEBPB may be the target gene of itraconazole to exert the therapeutic effect, and silencing CEBPB also inhibited glycolysis and tumor growth. These results provide a theoretical basis for understanding the new mechanism of energy metabolism and targeted/combined therapy in CRC.

AUTHOR CONTRIBUTIONS

Yong Zhang: Formal analysis; methodology; software; visualization; writing – original draft. Lu Li: Formal analysis; methodology; software; visualization; writing – original draft. Feifei Chu: Formal analysis; methodology; validation; writing – original draft. Huili Wu: Funding acquisition; resources; supervision; writing – review and editing. Xingguo Xiao: Data curation; methodology; software; writing – review and editing. Jianping Ye: Conceptualization; investigation; methodology; resources; writing – review and editing. Kunkun Li: Conceptualization; investigation; project administration; supervision; writing – review and editing.

FUNDING INFORMATION

This work was supported in part by the Project of Natural Science Foundation of China (82303016), Henan Provincial Medical Science and Technology Research Project (SBGJ202103102), the Key Scientific Research Project of the University in Henan Province (23A310027), the Research Start‐up Fund Project of Zhengzhou Central Hospital (KYQDJJ2021003).

CONFLICT OF INTEREST STATEMENT

The authors declare that there are no conflicts of interest.

ETHICS STATEMENT

Approval of the research protocol by an Institutional Reviewer Board: this study was approved by the Ethics Committee of Zhengzhou Central Hospital affiliated with Zhengzhou University.

Informed Consent: All samples were collected on condition of informed consent.

Registry and the Registration No. of the study/trial: N/A.

Animal Studies: All animal experiments in this study were performed under the institutional ethical guidelines approved by the Ethics Committee of Zhengzhou Central Hospital affiliated with Zhengzhou University.

Supporting information

Appendix S1.

CAS-115-1154-s001.docx (966.9KB, docx)

ACKNOWLEDGMENTS

Not applicable.

Zhang Y, Li L, Chu F, et al. Itraconazole inhibits tumor growth via CEBPB‐mediated glycolysis in colorectal cancer. Cancer Sci. 2024;115:1154‐1169. doi: 10.1111/cas.16082

Yong Zhang and Lu Li equal contribution and co‐first authors.

Contributor Information

Jianping Ye, Email: yejianping@zzu.edu.cn.

Kunkun Li, Email: likunkun770610@zzu.edu.cn.

DATA AVAILABILITY STATEMENT

The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in the National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA012185) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Appendix S1.

CAS-115-1154-s001.docx (966.9KB, docx)

Data Availability Statement

The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in the National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA012185) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa.

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