Background
Bladder cancer (BLCA) is a heterogeneous malignant tumor [
1]. There are about 500,000 new cases every year worldwide, posing a serious threat to human health [
2]. To date, a variety of therapeutic methods, including surgical resection, chemotherapy, radiotherapy and immunotherapy, have been widely used in the treatment of BLCA [
3,
4]. However, the overall curative effect is still not ideal enough to achieve a radical cure. Hence, it is of great clinical significance to make clear the mechanisms of cancer occurrence and progression, and to screen the pivotal factors for the early diagnosis and targeted therapy for BLCA.
Known as the Warburg effect, tumor cells preferentially choose the glycolytic pathway to provide energy for cell growth, even under aerobic conditions, rather than the more productive oxidative phosphorylation pathway [
5,
6]. As the core energy metabolism characteristics of solid malignant tumors, Warburg effect is characterized by active glycolysis pathway, decreased oxygen consumption, increased glucose uptake rate, but less ATP production and significantly increased lactic acid production in metabolites [
6]. Increasing studies have demonstrated that glycolytic enzymes such as enolases, play a pivotal role in cancer development [
7‐
9]. Enolase is an essential enzyme in the process of glycolysis, catalyzing 2-phosphoglycerate into phosphoenolpyruvate [
10,
11]. The three isoforms of ENO in mammalian cells include: ENO1, which is widely present in a variety of tissues [
12,
13]; ENO2, which is mainly expressed in neurons and neuroendocrine tissues [
12,
14]; and ENO3, which is expressed in muscle tissues [
15,
16].
ENOs are multifunctional molecules that not only participate in glycolysis pathway and regulate energy metabolism homeostasis, but also have a hand in the occurrence and development of various tumors [
17]. Recent reports have demonstrated that ENO1 plays a critical role in various tumor progression [
18‐
22]. For example, ENO1 was overexpressed in gastric cancer and functioned as a potential carcinogen to promote tumor progression [
23]. In addition, ENO2 was reported to be upregulated in BRAF V600E-mutated colorectal cancer and promote cells proliferation and migration [
24]. In another study, ENO3 was identified as an effective clinical biomarker for its selective role in the development of targeted therapies against lung adenocarcinoma [
25]. These findings indicated that the ENO isoforms have significant value in different types of cancer. Nonetheless, the association between ENO isoforms and the prognosis of patients with BLCA is rarely reported.
Herein, we utilized bioinformatics analysis tools to explore the expression and multilevel clinical value of ENOs in BLCA, and identified ENO1 as a promising immune-related target, providing a novel strategy for the diagnosis and clinical treatment of BLCA.
Methods
Expression of ENOs in BLCA
Clinical samples
Fresh BLCA tissues and adjacent non-tumorous tissues were acquired from the patients at the time of surgery from the Shanghai General Hospital. Formalin-fixed, paraffin-embedded BLCA tissues and correlative clinicopathological information were also collected from Shanghai General Hospital.
Cell culture and transfection
The human BLCA cell lines 5637 and UMUC-3 were cultured in RPMI-1640 medium (Invitrogen) at 37 °C with 5% CO
2. All media are supplemented with 10% FBS and penicillin/streptomycin. For the knockdown assay, small interfering RNAs targeting ENO1 (siENO1-1 and siENO1-2) were applied, and scramble siRNAs (siNC) as the negative control. The siRNA sequences targeting ENO1 were listed in Additional file
1: Table S1.
RNA isolation and quantitative Real-Time PCR
Total RNA was isolated from the cultured cells using the TRIzol reagent (TaKaRa), RNA was then converted into cDNA by applying the Prime-Script RT-PCR kit (TaKaRa). The mRNA expression levels of genes were examined using SYBR Green in an ABI 7500 StepOne Plus Real-Time PCR instrument (Applied Biosystem). The specific primer sequences were listed in Additional file
1: Table S1.
Western blotting and immunohistochemistry
Western blotting was performed according to the standard methods as previously described [
26]. Primary antibodies against ENO1 (1:2000, 11,204–1-AP, Proteintech) and GAPDH (1:1000, #5174, Cell Signaling Technology) were used. IHC staining of paraffin-embedded tissues with antibody against ENO1 (1:100, 11,204–1-AP, Proteintech) was performed following the standard procedures as previously described [
27].
Enrichment analysis of ENO1 co-expression network in BLCA
The stat packet of R software was employed to determine the co-expression genes associated with ENO1 expression in TCGA-BLCA. The clusterProfiler package of R software was utilized to perform GO function and KEGG pathway enrichment analysis of co-expressed genes.
Gene set enrichment analysis
GSEA v4.1.0 software was applied to perform GSEA to investigate meaningful biological processes associated with ENO1 expression. Pathways with nominal p-value < 0.05 and FDR < 0.25 were considered significantly enriched.
Prognostic analysis
Survival analysis was performed using the survival package and survminer package of R software to draw the Kaplan–Meier curves on BLCA samples. Survival package was also applied to perform univariate and multivariate Cox analysis, and survivalROC package was utilized to draw ROC curves.
Cell proliferation assay
2,000 cells/well were seeded into a 96-well plate. If cells adhered to the bottom, 10 μL MTT was added to each well for 4 h at 37 °C and it was identified as 0 h. The formazan crystals were dissolved in dimethyl sulfoxide (DMSO) at 37 °C for 15 min and the absorbance at 490 nm was examined. After 24, 48, and 72 h, the similar procedure was performed.
Transwell invasion assay
A total of 1 × 105 cells were seeded into the top of an 8 μm pore-size Transwell chamber pre-coated with diluted Matrigel (BD Biosciences), then 500 μL medium containing 10% FBS was added to the bottom chamber. After the incubation for 24 h, cells were fixed in formaldehyde, stained with crystal violet, and counted by applying a microscope.
Immune evaluation
CIBERSORT package of R software was used to detect the proportion of 22 immune cells in BLCA samples with low and high ENO1 expression, and Pearson’s correlation was assessed between the proportions and ENO1 expression.
Discussion
Bladder cancer (BLCA) is one of the most common malignancies in the genitourinary system [
2]. Its high incidence and recurrence rate exhort us to excavate novel biomarkers and therapeutic targets for early diagnosis and treatment [
32]. It is widely accepted that aerobic glycolysis is the main way of tumor cell productivity [
33]. Therefore, it is possible to inhibit tumor cell aerobic glycolysis by prohibiting the activity of pivotal glycolysis enzymes, thereby suppressing tumor cell proliferation and metastasis. Enolase is the crucial enzyme in the glycolysis pathway, catalyzing the conversion of 2-phosphoglycerate to phosphoenolpyruvate [
10,
11]. Therefore, interference with enolase may inhibit the growth of tumor cell by inhibiting the glycolytic pathway, suggesting that enolase has the potential value as therapeutic target.
Hence, to demonstrate the potential worth of ENOs in BLCA, the expression and clinical prognostic value of ENOs were analyzed. First of all, we were pleasantly surprised to find that the expression level of ENO1, but not other ENO isoforms, was significantly up-regulated at the mRNA level in BLCA in Oncomine, TIMER, UALCAN, TCGA-BLCA and GSE13507 databases. Western blotting and immunohistochemical further demonstrated aberrant overexpression of ENO1 at the protein level. Subsequently, the clinical prognostic value of ENO1 was explored. High expression of ENO1 was prominently correlated with high pathological grade and advanced clinical stage. Moreover, overexpression of ENO1 predicted worse prognosis in patients with BLCA. ROC curves also showed that ENO1 had significant diagnostic value for BLCA. Meanwhile, nomogram model illustrated that ENO1 could serve as an independent prognostic factor, which could be utilized to estimate the prognosis of patients. Of particular note, few studies have also elevated the circulating level of ENO1 in cancer patients. For example, ENO1 was overexpressed in the plasma of patients with pancreatic cancer, and the increased plasma ENO1 level was correlated with prognosis and disease progression [
34]. In addition, abnormally high circulating ENO1 levels have also been reported in non-small cell lung cancer [
35]. Interestingly, plasma ENO1 levels decreased progressively in normal, precancerous condition of the esophagus and esophageal cancer, exactly in contrast to the tissue expression of the protein [
36]. These findings implied us further study should be attached to detect the plasma ENO1 level in BLCA patients, which will be of great significance for the translational application of ENO1 in the diagnosis and treatment of BLCA.
The current research on the function of ENO1 in tumor is primarily focused on its effects in glycolysis, while comprehensive analysis of ENO1 in BLCA is less studied. We first identified genes that were significantly correlated with ENO1by constructing a co-expression network, among which TPI1, RAN and GAPDH showed the strongest correlation with ENO1 in BLCA. Previous studies have demonstrated that TPI1 and RAN exerted crucial effects on tumor initiation and progression [
37‐
40]. For example, the reduction of TPI1 in extracellular vesicles mediated by Rab20 downregulation facilitates aerobic glycolysis to drive hepatocarcinogenesis [
41]. RAN promotes membrane targeting and stabilization of RhoA to enhance ovarian cancer cell growth and invasiveness [
42]. While the function of TPI1 and RAN in BLCA has not been elucidated. Considering the strong correlation of TPI1, RAN with ENO1, and the significant value of ENO1 in BLCA, the role of TPI1 and RAN in BLCA deserves our attention and further exploration. The GO and KEGG function enrichment analyses based on the co-expression network revealed that ENO1 was also involved in many other vital pathways, such as cell cycle and immune-related processes, in addition to regulating glucose metabolism. These findings were consistent with the results of GSEA presented in this study, further reinforcing the effects of ENO1 in regulating cell cycle and immune activity. Additionally, Function experiments demonstrated that ENO1 depletion inhibited cancer cell aggressiveness, further indicating that ENO1 functions as a bad prognostic factor in BLCA.
Increasing evidence has demonstrated that infiltrating immune cells in the tumor microenvironment plays a crucial role in tumorigenesis and progression, thereby affecting the prognosis of tumor patients [
43‐
45]. In this study, we reported that ENO1 expression was significantly correlated with the infiltration of activated memory CD4 cells, resting NK cells, M0 macrophages, neutrophils, naive B cells, regulatory T cells, monocytes, and resting mast cells in BLCA. Moreover, we also identified that ENO1 was involved in multiple immune-related processes, suggesting that ENO1 might exert important regulatory effects in immune-related pathways. Together, these findings indicated that ENO1 might function as a crucial regulator in tumor immunity, as well as a potential biomarker associated with immune infiltration in BLCA. However, the mechanisms involved in how ENO1 affects immune cell infiltration have not been fully elucidated, further in-depth investigation is required to be carried out to elucidate the exact function of ENO1 in the tumor-immune microenvironment.
Changes in tumor metabolism provide new therapeutic targets for tumor therapy. As mentioned earlier, genes related to the glycolytic pathway have been the focus of tumor target research. As the core catalytic enzyme of glycolysis, increasing studies have begun to look for molecules to effectively inhibit ENO1 activity. Previous studies have found that PhAH was a pan-enolase inhibitor, which could effectively inhibit the activity of ENO1, thereby suppressing the growth of pancreatic, breast, and lung cancers [
17,
46]. Another study reported that the small molecule AP-III-a4 could directly bind to ENO1 and repress its catalytic activity, thereby prohibiting tumor cell survival without cytotoxicity to normal cells [
18,
47]. Taken together, the above research findings and our experimental results have significant guiding help for our clinical research in the future, suggesting that we can manufacture effective inhibitors specific to ENO1 in the future, develop a new tumor treatment strategy for ENO1, and apply to the clinic, which will provide new methods and strategies for clinical treatment of BLCA and even other tumors.
Although the present study initially revealed the association of ENO1 with BLCA, some limitations still exist. Firstly, we confirmed abnormally high ENO1 expression in BLCA at both mRNA and protein levels. However, further studies should be conducted to investigate the specific role and potential molecular mechanisms of ENO1 in tumorigenesis, progression, and immune infiltration. As a metabolite of ENO1 during glycolysis, the biological role of phosphoenolpyruvate in BLCA has also not been fully evaluated, enlightening us that further exploration should be attached to the biological significance of the metabolites correlated with ENO1. In addition, most of the analyses were performed based on TCGA and GEO cohorts, which lack further experimental validation. More in-depth exploration to explain these findings should be systematically interpreted in vitro and in vivo to make the results more convincing.
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