PINK1 suppresses malignant phenotypes in esophageal squamous cell carcinoma

Abstract

Objective

Esophageal squamous cell carcinoma (ESCC) is a common tumor characterized by a poor prognosis. PINK1 is strongly associated with tumorigenesis. However, the role of PINK1 in the progression of ESCC has not been elucidated.

Methods

The expression levels of PINK1 in tumor samples and corresponding normal tissues were evaluated using RNA-sequencing and gene expression array datasets. Pathway enrichment and immune infiltration analyses were performed to explore the role of PINK1 in ESCC development. Subsequently, cell counting kit-8, colony-forming assay, and Transwell assays were implemented to measure the proliferation and motility of ESCC cells. The glucose, ATP, pyruvate, and lactate concentrations were measured using suitable assay kits.

Results

PINK1 expression was significantly lower in ESCC samples across independent cohorts. In vitro assays demonstrated that PINK1 could inhibit the proliferation, migration, and invasive capabilities of ESCC cells. Furthermore, PINK1 could decrease intracellular glucose, lactic acid, pyruvic acid, and ATP levels in ESCC cells, whereas the glycolytic inhibitor 2-DG could abrogate its effect. Additionally, immunosuppressive-related gene sets were enriched in the PINK1 low-expression group. Immune infiltration analysis revealed that PINK1 expression was positively correlated with dendritic cells and T helper 1 cells within the tumor microenvironment.

Conclusions

PINK1 inhibits cell growth, movement, glycolysis, and immune activation in ESCC, making it a promising therapeutic target.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12957-025-03879-y.

Introduction

Globally, esophageal carcinoma (ESCA) is a prevalent malignancy with exceedingly high incidence and mortality rates, particularly in China [1]. Esophageal squamous cell carcinoma (ESCC), which constitutes over 90% of all esophageal carcinoma cases [2], has seen a slight improvement in the 5-year survival rate among patients with advancements in diagnostic modalities and multimodal therapy in recent decades [3, 4]. It is imperative to investigate the factors involved in the development and progression of ESCC and their clinical implications for accurately assessing patient prognosis and delivering appropriate treatment.

PTEN Induced Kinase 1 (PINK1), initially identified in 2001, belongs to the family of genes associated with Parkinson’s disease (PD) and plays a crucial role in the pathogenesis and progression of PD [5]. Composed of 581 amino acids, PINK1 influences cellular injury, inflammation, and tumorigenesis [6–8]. Research by Liu et al. [9] indicated that PINK1 can suppress the proliferation of liver cancer cells. In colorectal cancer, PINK1 expression is downregulated, and the loss of PINK1 expression is correlated with an increased incidence of colon tumors [10]. Additionally, PINK1 has been demonstrated to promote pancreatic tumor formation by influencing mitochondrial iron accumulation [11]. Nonetheless, the precise function of PINK1 in the development of ESCC has yet to be determined.

In this study, we evaluated the expression levels of PINK1 in ESCC from multiple independent cohorts. Subsequent pathway enrichment and immune infiltration analyses were performed to explore the role of PINK1 in the development of ESCC. Furthermore, in vitro assays were performed to analyze the impact of PINK1 overexpression and knockdown on the proliferation, migration, invasion, and glucose metabolism of ESCC cells, highlighting the potential of PINK1 as a therapeutic target.

Materials and methods

Bioinformatics analysis

The expression profile of the PINK1 gene in pan-cancer and normal samples was sourced from The Cancer Genome Atlas (TCGA). The RNA-seq data of unpaired and paired samples in ESCA were also collected and processed from TCGA. GSE23400 data from the GEO database were standardized and visualized by the R (4.2.1) and ggplot2 [3.3.6] packages. The Wilcoxon rank-sum test was employed for a differential expression analysis of PINK1 across diverse cancer types. The Shapiro-Wilk test was applied to evaluate the normality of the data. Subsequently, a Wilcoxon rank-sum test was conducted for statistical analysis, deemed significant at a P < 0.05.

ESCC patients from TCGA database were categorized into groups according to high and low expression levels of PINK1. The R package DESeq2 was employed to analyze differentially expressed genes between these two groups for Gene Set Enrichment Analysis [GSEA] with gene signatures sourced from the GSEA Molecular Signature Database (c2.all.v2022.1.Hs.symbols.gmt[Curated/Pathway]). The significance threshold was established with a false discovery rate (FDR) < 0.25 and p.adjust < 0.05.

The levels of immune infiltration were estimated in both PINK1-low and PINK1-high groups using the single-sample Gene Set Enrichment Analysis (ssGSEA) method. Spearman correlation was calculated between PINK1 levels and the levels of immune cell infiltration. The statistical significance threshold was defined as a P-value < 0.05.

Cell culture

TE-1 (TCHu89) and KYSE-150 (TCHu236) cells were obtained from the Cell Bank, Type Culture Collection of Chinese Academy of Sciences. The cells were cultured in a complete medium, which comprised the Roswell Park Memorial Institute (RPMI) 1640 (Gibco, USA), 10% fetal bovine serum (FBS; OriCell, China), and 1% antibiotics containing penicillin and streptomycin (Beijing Solarbio Science & Technology Co., Ltd., China), and were incubated at 37 °C and 5% CO₂.

Cell transfection

PINK1 plasmid and empty vector (GenePharma, China) were transfected into TE-1 cells using Lipofectamine 3000 and P3000 (Invitrogen, USA). The PINK1 interference lentivirus (GeneChem, China) was used to infect KYSE-150 cells for 24 h. Stable KYSE-150 cells with PINK1 knockdown were subsequently screened with puromycin at a concentration of 2.0 µg/mL. Quantitative real-time PCR (qRT-PCR) and Western blotting were employed to assess the expression level of PINK1 in TE-1 cells and KYSE-150 cells.

qRT-PCR

Total RNA was isolated using the E.Z.N.A. Total RNA Kit I per the manufacturer’s guidelines (R6834-01, Omega Bio-tek, USA). Reverse transcription was conducted using the Revert Aid First Strand cDNA Synthesis Kit (K1622, ThermoFisher Scientific, USA). After that, qRT-PCR was conducted using TB Green Premix Ex Taq II (RR820A, Takara Biomedical Technology, Japan) on Real-Time PCR System (CFX96, Bio-Rad, USA). Relative expression levels were assessed by normalizing the expression level of each target to ACTB, and relative mRNA fold changes were calculated using the 2^−ΔΔCt method. The primer sequences for PINK1 were 5’-CGAGAGGCCAGCAAGAGAC-3’ and 5’-CCGATTGTTGGAGGAGCCAG-3’; for ACTB: 5’-AACCGCGAGAAGATGACCCAG-3’ and 5’-GGATAGCACAGCCTGGATAGCAA-3’.

Western blotting

Proteins were extracted from cells, and the protein lysates were separated using a 8% resolving gel and a 5% stacking gel before being transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skimmed milk powder in tris-buffered saline (TBS) and incubated with PINK1 (sc-517353; 1:1000, Santa Cruz Biotechnology, USA) and β-actin (TA-09; 1:1000, Zsbio, China) antibodies overnight at 4 °C. On a subsequent day, the membrane was washed with TBS, incubated with horseradish peroxidase-labeled secondary antibodies for 2 h at room temperature, and then rinsed with TBS again. Finally, immunoblots were visualized using a Western Luminescent Detection kit (BLA520A, Biosharp), and the images were captured with a chemiluminescence imager.

Cell counting Kit-8 (CCK-8) assay

A total of 5 × 10³ cells were inoculated into 96-well plates and incubated at 37 °C for the designated duration. Following this incubation, the Cell Counting Kit-8 (CCK-8) (BS350B, Biosharp, China) was added and incubated for two hours at 37 °C. Subsequently, the optical density (OD) at 450 nm was measured using a microplate reader.

Colony-forming assay

After transfection, 3 × 10³ of KYSE-150 cells were seeded in each well of a 6-well plate. Ten days later, the cells were washed twice with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 30 min, and stained with 1% crystal violet for 20 min. Finally, cell colonies were counted for statistics.

Transwell assay

Cells were suspended in 200 µL of RPMI 1640 and positioned within the upper chambers, featuring an 8 μm pore size (Corning, USA). Subsequently, 650 µL of RPMI 1640 containing 10% FBS was dispensed into the bottom chamber. Following a 24-hour incubation period at 37℃, the cells traversed through the upper Transwell chambers were subjected to three washes with PBS. Subsequently, the cells were fixed using 4% ice-cold paraformaldehyde and stained with a 0.1% solution of crystal violet. In the end, stained cells were counted with an inverted fluorescence microscope (CX23, Olympus, Japan). For the invasion assay, matrix gel was introduced to the upper chamber, and cells were incubated for 48 h at 37℃. The other procedure was identical to the migration assay.

Detection of glycolysis

Cells were inoculated into 6-well plates and incubated with the complete medium at 37℃ for 48 h. For the KYSE-150 cells treated with 2-Deoxy-D-glucose (2-DG), cells were inoculated and cultured in a complete medium at 37℃ for 24 h until they adhered to the surface, then 20 mM of 2-DG (HY-13966, MCE, USA) was added, and the cells were further cultured for 24 h. Cells were then collected and centrifuged, and the supernatant was discarded. Cells are ready for use after cell lysis treatment. The concentration of glucose, ATP, pyruvate, and lactate was determined using the Glucose Assay Kit (E1011, APPLYGEN, China), ATP Assay Kit (S0026B, Beyotime, China), Pyruvate Assay Kit (A081-1-1, Nanjing Jiancheng, China), and Lactic Acid Assay Kit (A019-2-1, Nanjing Jiancheng, China), following the manufacturer’s instructions.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software (v 10.1.2). An unpaired t-test or ANOVA was employed to conduct a comparative evaluation between groups. Statistical significance was set at P < 0.05.

Results

PINK1 expression is downregulated in ESCC

Initially, a Pan-Cancer analysis of PINK1 within TCGA demonstrated that it generally has low levels of expression across various malignancies, such as in head and neck squamous cell carcinoma (HNSC), bladder cancer (BLCA), and esophageal carcinoma (ESCA) (Fig. 1A). In paired pan-cancer samples, PINK1 expression was decreased in most tumor types, including ESCA (Fig. 1B). Tumor tissues exhibited significantly lower expression levels of PINK1 compared to normal tissues in unpaired samples from TCGA-ESCC dataset (Fig. 1C). Moreover, in unpaired and paired samples from the GSE23400 dataset, PINK1 levels were notably lower in tumor tissues than in normal esophageal tissues (Fig. 1D, E). Overall, these findings suggest that PINK1 expression is downregulated in human ESCC.

Fig. 1.

Low expression levels of PINK1 in ESCC

(A) PINK1 expression in unpaired samples from TCGA datasets. (B) PINK1 expression in paired samples from TCGA datasets. (C) Expression of PINK1 in unpaired samples obtained from TCGA-ESCC dataset. (D, E) Expression levels of PINK1 in the unpaired (D) and paired (E) samples from the GSE23400 dataset. ns, not significant; *P < 0.05, ** P < 0.01, ***P < 0.001

PINK1 inhibits proliferation of ESCC cells

Our previous study [12] found that PINK low expression was enriched in the cell cycle, DNA replication, and RNA polymerase pathways. To clarify the function of PINK1 in ESCC, we achieved and confirmed the knockdown of PINK1 expression (Fig. 2A). The results from the CCK-8 assay indicated that KYSE-150 cell proliferation was significantly enhanced in the PINK1 down-regulation group (Fig. 2B). In the colony-forming assay, the number of colonies of ESCC cells with PINK1 knockdown was more than that of the control group (Fig. 2C). On the contrary, TE-1 cell proliferation was suppressed in the PINK1 overexpression group (Fig. 2D, E). Taken together, PINK1 can inhibit the proliferation of ESCC cells.

Fig. 2.

PINK1 inhibits the proliferation of ESCC cells

(A) Knockdown of PINK1 expression in KYSE-150 cells confirmed by qRT-PCR and western blotting. (B) Knockdown of PINK1 expression in KYSE-150 cells significantly enhanced cell growth. (C) The number of colonies in the group with PINK1 knockdown was more than in the control group. (D) Overexpression of PINK1 in TE-1 confirmed by qRT-PCR. (E) Growth of TE-1 cells was inhibited following PINK1 overexpression. *P < 0.05, ** P < 0.01, ***P < 0.001

PINK1 inhibits motility of ESCC cells

To further explore the function of PINK1 in ESCC, a functional enrichment analysis of PINK1 was performed. According to levels of PINK1 expression, TCGA-ESCC patients were categorized into low and high-expression groups. The ALONSO_METASTASIS_EMT_UP gene set, closely related to cancer metastasis [13], was enriched in the PINK1 low-expression group (Fig. 3A). To ascertain the influence of PINK1 on the motility of ESCC cells, the migration and invasion capacity of TE-1 and KYSE-150 cells were assessed through Transwell assays. The results showed that TE-1 cells with PINK1 overexpression exhibited a remarkably decreased number of migrating cells compared with the control group, and the number of invading cells was lower than in control cells (Fig. 3B). In the KYSE-150 cells with PINK1 down-regulation, the number of migrating and invading cells prominently increased compared with the control group (Fig. 3C). These findings suggest that PINK1 can suppress the motility of ESCC cells.

Fig. 3.

PINK1 inhibits the motility of ESCC cells

(A) GSEA functional enrichment analysis of the EMT-associated pathway. (B) Counts of migrating and invading TE-1 cells with overexpression of PINK1. (C) The number of migrating and invading KYSE-150 cells with PINK1 knockdown. *P < 0.05, ** P < 0.01, ***P < 0.001

PINK1 represses glycolysis

Mitochondria serve as the primary source of energy production in eukaryotic cells, with PINK1 playing a pivotal role in mitochondrial quality control and the regulation of energy generation processes [7, 8]. Glycolysis is one of the critical pathways for energy supply to tumor cells. The GSEA on TCGA-ESCC dataset showed that the glycogen metabolism pathway was enriched in the PINK1-low expression group (Fig. 4A). Consequently, we verified the impact of PINK1 on the glycolysis of ESCC cells. The findings indicated that the levels of intracellular glucose, pyruvate, lactic acid, and ATP in TE-1 cells with PINK1 overexpression were decreased (Figs. 4B‒E), suggesting that PINK1 can inhibit glycolysis in ESCC cells. In the PINK1 knockdown group, we observed a significant increase in intracellular glucose, pyruvate, lactate, and ATP levels than the control group. However, treatment with 2-DG, a glycolysis inhibitor, exhibited reduced levels of intracellular glucose, pyruvate, lactate, and ATP in KYSE-150 cells with PINK1 down-regulation (Figs. 4F‒I). These results manifest that PINK1 suppresses the glycolysis process in ESCC cells.

Fig. 4.

PINK1 represses glycolysis

(A) The glycogen metabolism pathway was closely related to PINK1 low expression. (B‒E) Intracellular glucose, pyruvate, lactic acid, and ATP levels were decreased in TE-1 cells with PINK1 overexpression. (F‒J) Intracellular glucose, pyruvate, lactic acid, and ATP levels were measured in KYSE-150 cells with PINK1 knockdown and 2-DG treatment. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001

PINK1 promotes Anti-tumor immune response in ESCC

Some studies reported that PINK1 regulates innate and adaptive immunities [14–16]. Therefore, we employed GSEA to analyze the immune pathways associated with the level of PINK1 expression in TCGA-ESCC dataset. Results showed that PD-1 signaling, nef-mediated downregulation of MHC class I complex cell surface expression, CTLA4_pathway, which could play a role in suppressing immune responses and promoting immune evasion in tumors, were enriched in the PINK1 low expression group (Figs. 5A‒C). Furthermore, the levels of immune infiltration for the 24 types were examined in PINK1 low- and high-expression groups by the ssGSEA method in TCGA-ESCC dataset. The results demonstrated that PINK1 expression is positively correlated with immune cells such as cytotoxic cells, dendritic cells (DCs), and T helper 1 (Th1) cells (Fig. 5D). A scatter plot was subsequently generated to illustrate the relationship between the infiltration scores of each immune cell and PINK1 expression (Fig. 5E), with significant correlations indicated in Figure S1. A correlation chord was created to illustrate the relationship between PINK1 expression and the infiltration scores of various immune cells (Fig. 5F). These findings suggest a robust correlation between high PINK1 expression and the immune activation status of ESCC.

Fig. 5.

Immune infiltration analysis of PINK1

(A‒C) GSEA of immune-related pathways. (D) Comparison of enrichment scores of 24 immune cells between PINK1-high and -low groups, presented as box plots. (E) The lollipop plot depicts the relationship between 24 immune cells and PINK1 expression. (F) The chord diagram visualizes the correlations, utilizing blue lines to signify negative correlations and pink lines for positive correlations. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001

Discussion

The family of genes associated with Parkinson’s disease (PD) plays a significant role in the development and progression of tumors by modulating cell proliferation and migration [6, 17, 18]. PINK1, an essential member of this gene family, is extensively expressed in healthy tissues and different types of tumors. Nevertheless, the role of PINK1 in the development and progression of ESCC has been ambiguous. This study found that PINK1 expression was decreased across several tumor types, including ESCC. This finding was corroborated in multiple datasets of ESCC. Studies have reported that PINK1 deficiency can stimulate cell proliferation and migration in gastric cancer [19]. In human colorectal tumors, PINK1 is downregulated, and its overexpression induces apoptotic cell death, thereby suppressing tumor cell growth [10]. Conversely, upregulated PINK1 can accelerate cell proliferation in breast cancer [20]. Furthermore, PINK1 has been identified as a direct treatment target for the combined treatment of metformin and arsenic trioxide in cervical cancer [21]. The findings above imply that PINK1 holds potential as a promising candidate for therapeutic targeting in the treatment of ESCC.

Studies have shown that low expression of PINK1 in cancer cells can drive proliferation, invasion, and migration [10, 11, 20]. PINK1 suppresses the growth of glioblastoma cells by attenuating ROS and FOXO3a to regulate the Warburg effect [22]. PINK1 inhibits pancreatic cancer induced by mutant KRAS through a HIF-1α dependent Warburg effect [11]. A comprehensive Pan-Cancer analysis of PINK1, conducted using data from TCGA, revealed that it generally exhibits low levels of expression in various types of cancer, including ESCC. Functional enrichment analysis was performed to gain deeper insight into the role of PINK1 in the molecular mechanisms driving the growth of ESCC. The findings suggest that PINK1 low expression plays a role in regulating EMT. Therefore, we propose that PINK1 deficiency may accelerate the progression of ESCC. The up-regulated PINK1 expression impaired the proliferation, migration, and invasion capabilities of ESCC cells. Knocking down PINK1 expression exhibited the opposite results. This confirms that PINK1 inhibits the growth and metastasis of ESCC.

PINK1 is crucial for preserving mitochondrial function and ensuring quality control within the cell. Mitochondria, as crucial cellular organelles and power generators [23], play a significant role in energy metabolism and cell homeostasis [24, 25]. GSEA exhibited that the glycogen metabolism pathway was enriched in the PINK1 low-expression group. Furthermore, ESCC cells were overexpressed or knocked down PINK1 along with 2-DG treatment and the intracellular glucose content, pyruvate, lactic acid, and ATP levels were measured. The results revealed that PINK1 can inhibit glycolysis in ESCC. Overexpressed PINK1 suppresses the growth of glioblastoma cells via attenuating ROS and FOXO3a to regulate the Warburg effect [22]. The precision regulatory mechanisms of PINK1 regulating the development of ESCC by modulating mitochondrial function and glycolysis will be one of our focal points for future exploration.

Research has demonstrated that the role of PINK1 in immune regulation involves various aspects, including regulating inflammatory responses, affecting T cell function, interacting with inflammasomes, and modulating dendritic cell function [15, 26, 27]. GSEA revealed that PD-1 signaling, nef-mediated downregulation of MHC class I complex cell surface expression, and CTLA4_pathway were closely related to PINK1 low expression in ESCC patients. Further analysis of the correlation between PINK1 expression and immune cells demonstrated a positive correlation with immune cells such as Th1 cells, dendritic cells (DCs), and cytotoxic cells. Th1 cells are essential in coordinating anti-tumor immunity and can induce cytotoxic cells to eliminate tumor cells [28, 29]. DCs require PINK1-mediated phosphorylation of BCKDE1α to promote fatty acid oxidation to support immune function. DCs play a role in anti-tumor immunity by activating cytotoxic T lymphocytes to specifically kill tumor cells, presenting tumor-associated antigens [30]. Given that PINK1 positively correlates with Th1 cells and DCs, it may promote therapeutic effects on tumors. These findings suggest that PINK1 expression benefits the tumor immune response and boosts the effectiveness of tumor immunotherapy.

Conclusions

PINK1 is expressed at low levels in ESCC, and the interactions and pathways identified offer significant insights into its role in ESCC development. PINK1 can suppress the proliferation, motility, and glycolysis of ESCC cells. As a tumor suppressor, PINK1 is likely implicated in immune response and glycolysis, holding promise as a potential therapeutic target to enhance the prognosis of patients with ESCC. Nonetheless, this study has certain limitations. Although it provides significant preliminary findings, these results need to be validated in animal models and a broader patient population. Additionally, the molecular mechanisms that PINK1 regulates immune response and glycolysis were not specifically elucidated and warrant further exploration.

Electronic supplementary material

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Abbreviations

ESCC

Esophageal Squamous Cell Carcinoma

ESCA

Esophageal Carcinoma

PD

Parkinson’s Disease

TCGA

The Cancer Genome Atlas

GSEA

Gene Set Enrichment Analysis

FDR

False Discovery Rate

ssGSEA

Single-sample Gene Set Enrichment Analysis

RPMI

Roswell Park Memorial Institute

qRT-PCR

Quantitative Real-time PCR

PVDF

Polyvinylidene Fluoride

2-DG

2-Deoxy-D-glucose

HNSC

Head and Neck Squamous Cell Carcinoma

Author contributions

Xiangyun Lu, Yongkun Yao, and Lianghai Wang carried out the conception and design of the study. Yuhui Pei conducted the experiments, while Hao Peng, Man Li, and Jing Li analyzed the expression data. Xiangyun Lu drafted the manuscript, while Lianghai Wang contributed significant revisions for essential intellectual content.

Funding

This research was supported by the Bingtuan Science and Technology Program (2022ZD002) and the Science and Technology Program of Shihezi University (ZZZC202133, ZZZC201960A, RCZK202450, and ZZZC2023023).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.