Expression of Autophagy-Related Proteins Beclin-1 and LC3-Ⅱ in Lung Cancer Metastatic Pleural Effusion

YAO Wenjing¹, WANG Cuifeng²*, GAO Jinliang³, REN Meiying², JING Xuefen², FU Yuhua²

¹Baotou Medical College, Inner Mongolia University of Science and Technology, Baotou 014040, China
²Department of Clinical Laboratory, The First Affiliated Hospital of Baotou Medical College, Baotou 014010, China
³Laboratory of Molecular Medicine, Ordos Central Hospital, Ordos 017000, China

Corresponding author: Wang Cuifeng, Chief technician; E-mail: wangcuifeng1973@vip.sina.com

Abstract

Background: Malignant pleural effusion represents a critical clinical indicator of advanced-stage malignancy and metastatic progression. Current diagnostic methods for determining pleural effusion characteristics suffer from significant limitations, preventing timely and accurate assessment. While metastatic lung cancer constitutes the primary etiology of malignant pleural effusion, the expression patterns and diagnostic potential of the autophagy nucleation key protein Beclin-1 and LC3-Ⅱ—a marker for monitoring cellular autophagy levels—remain unreported in metastatic pleural effusion, necessitating further investigation.

Objective: To analyze the expression of autophagy-related proteins Beclin-1 and microtubule-associated protein 1 light chain 3-Ⅱ (MAP1LC3-Ⅱ) in lung cancer-associated malignant pleural effusion and evaluate their potential value in early clinical diagnosis.

Methods: From May 2022 to July 2023, bioinformatics analysis of Beclin-1 and LC3 was performed using GEO, GEPIA2, and GeneMANIA databases. Pleural effusion samples were classified into malignant (n=95) and benign (n=190) groups based on liquid-based cytology and clinical data. RT-PCR and Western blot were used to quantify mRNA and protein expression levels of Beclin-1 and LC3-Ⅱ, respectively, with immunofluorescence assay validating LC3 puncta formation.

Results: Bioinformatics analysis confirmed that both Beclin-1 and LC3 were present in lung cancer differentially expressed gene datasets, showing differential expression between tumor and normal tissues. RT-PCR revealed significantly higher mRNA expression of both Beclin-1 and LC3 in benign controls versus malignant cases (P<0.05). Western blot analysis demonstrated elevated Beclin-1 and LC3-Ⅱ protein abundance in benign specimens compared to the malignant group (P<0.05). Immunofluorescence microscopy identified increased FITC-labeled LC3-Ⅱ puncta, representing autophagosome quantity, in benign controls relative to malignant cases.

Conclusion: Analysis of expression differences in autophagy-related proteins Beclin-1 and LC3-Ⅱ between benign pleural effusion and lung cancer metastatic malignant pleural effusion may facilitate early diagnosis of malignant pleural effusion, providing novel perspectives for differential diagnosis and targeted therapeutic strategies.

Keywords: Lung neoplasms; Pleural effusion; Autophagy; Beclin-1; LC3-Ⅱ

Introduction

Lung cancer is one of the most common malignant tumors and constitutes a major cause of cancer-related mortality worldwide, with a five-year survival rate of only 3%. The vast majority of patients are diagnosed with advanced-stage disease or distant metastasis at initial presentation. Although novel therapeutic approaches have improved survival outcomes for lung cancer patients in recent years, prognosis remains unsatisfactory. Malignant pleural effusion (MPE) caused by lung cancer accounts for 50%~65% of all MPE cases and represents an important indicator of advanced disease and metastasis. Current diagnostic methods for pleural effusion, including routine examinations and biochemical analyses, lack specificity, while diagnostic markers demonstrate insufficient sensitivity. Cytomorphological assessment is further confounded by the nature of the effusion itself and the technical expertise of examiners, making accurate classification challenging and significantly impacting clinical decision-making. Therefore, investigating the mechanisms underlying MPE formation and identifying highly specific diagnostic markers is of critical clinical importance for precise early diagnosis and treatment planning.

Autophagy is a lysosomal degradation and recycling process of cytoplasmic proteins and damaged organelles regulated by autophagy-related genes (ATG). Numerous studies have demonstrated that autophagy plays a crucial role in tumor initiation, progression, and metastasis. Consequently, examining autophagy-related protein expression in lung cancer metastatic pleural effusion may represent an effective strategy for elucidating autophagy's mechanistic role in lung cancer and diagnosing effusion characteristics. Beclin-1 serves a critical function in autophagy initiation and regulation and functions as an autophagy marker. Microtubule-associated protein 1 light chain 3 (LC3) is a key downstream factor regulated by Beclin-1. The cytosolic form LC3-Ⅰ is conjugated to phosphatidylethanolamine, modifying it into the membrane-bound LC3-Ⅱ. LC3-Ⅱ activity positively correlates with cellular autophagy levels, effectively reflecting autophagic activity. However, the role of Beclin-1 and LC3-Ⅱ in lung cancer metastatic pleural effusion remains unreported. This study utilizes minimally invasive, readily obtainable pleural effusion specimens to investigate expression levels of Beclin-1 and LC3-Ⅱ, providing novel molecular evidence for distinguishing benign from malignant effusions.

Methods

1.1.1 GeneMANIA Database Analysis of Beclin-1 and LC3 Interacting Genes

From May 2022 to July 2023, the GeneMANIA database was used to screen for interacting proteins based on physical interactions, co-expression, predictions, co-localization, pathway associations, genetic interactions, and shared protein domains, with network diagrams constructed accordingly. Beclin-1's primary functions include participation in autophagy, phosphatidylinositol-3-kinase complex formation, response to nutrient levels and starvation, cellular response to extracellular stimuli, autophagosome formation, and regulation of cell division. LC3 primarily participates in macroautophagy, autophagosome organization and formation, and response to cellular starvation and stimuli (Figure 1 [FIGURE:1]).

1.1.2 Bioinformatics Analysis Based on GEO Database

The National Center for Biotechnology Information's GEO public database was queried using "Lung cancer" and "adjacent normal lung tissue" as keywords to retrieve lung cancer-related gene expression microarray data. The GSE19188 dataset, based on platform GPL570, was selected for analysis. Predicted genes from expression profiles of 91 tumor samples and 65 adjacent normal lung tissue samples were processed. Differentially expressed genes were screened using adjusted P<0.05 as the threshold. The ggplot package in R was used to generate volcano plots (Figure 2 [FIGURE:2]), with both Beclin-1 and LC3 present in the differentially expressed gene dataset (Table 1 [TABLE:1]).

1.1.3 TCGA Database Analysis Using GEPIA2

The GEPIA2 online tool (gepia2.cancer-pku.cn) was used to analyze TCGA data (lung adenocarcinoma: 483 cases, normal: 347 cases; lung squamous cell carcinoma: 486 cases, normal: 338 cases) via the Box plot function in Expression DIY. Beclin-1 and LC3 were entered in the "Gene" field, with lung adenocarcinoma and lung squamous cell carcinoma selected in "Datasets Selection," and PDF box plots were generated.

1.2 Experimental Subjects

A total of 400 pleural effusion samples from untreated patients were collected at the Clinical Laboratory Cell Room of the First Affiliated Hospital of Baotou Medical College between 2018 and 2023. After applying inclusion and exclusion criteria, 285 cases were selected, comprising 190 benign controls and 95 malignant cases. This study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Baotou Medical College (Approval No. 2022(5)).

1.2.1 Inclusion Criteria

(1) Pleural effusion confirmed by chest X-ray, CT scan, or ultrasound, with samples obtained via thoracentesis. Benign controls were defined as effusions with no malignant cells or abnormal tumor markers in cytological/biochemical analysis and clinical diagnosis of benign disease. (2) Malignant cases were defined as effusions caused by metastatic lung cancer with cancer cells identified by cytology or pleural biopsy and confirmed by clinical pathological records.

1.2.2 Exclusion Criteria

(1) No definitive clinical diagnosis; (2) Pleural effusion due to other diseases; (3) History of surgery, chemotherapy, molecular targeted therapy, or other anti-tumor treatments.

1.3.1 Sample Collection and Processing

Qualified pleural effusion specimens (100 mL) were aliquoted into 5 tubes, mixed with 2 mL PBS, centrifuged at 2000 r/min for 10 min, and supernatant discarded. Precipitates were divided into 5 tubes: two stored at -80°C and three used immediately for liquid-based thin-layer slide preparation and staining.

1.3.2 Liquid-Based Cytology for Pleural Effusion Classification

EasyFix liquid-based cytology was used to prepare smears, fixed with 95% ethanol for 15 min, stained with Wright-Giemsa, and mounted. Microscopic examination revealed benign effusions containing predominantly mesothelial cells, lymphocytes, neutrophils, and phagocytes with normal morphology. Malignant effusions displayed clustered, irregularly shaped cells with increased volume, abundant basophilic cytoplasm containing dark blue vacuoles, increased nuclear-cytoplasmic ratio, uneven nuclear chromatin, prominent nucleoli, and irregular nuclear membrane thickening (Figure 3 [FIGURE:3]). Based on clinical-pathological data, the benign group comprised 190 cases (119 male, 71 female; mean age 74.4±10.4 years) and the malignant group 95 cases (54 male, 41 female; mean age 67.8±12 years).

1.3.3 Real-Time PCR for Gene Expression

Primer sequences are listed in Table 2 [TABLE:2]. Total RNA was extracted using RNAiso Plus, quantified, and reverse-transcribed to cDNA on ice. Reaction parameters: (1) 95°C, 30 s; (2) 95°C, 5 s; 60°C, 34 s (40 cycles); (3) 95°C, 15 s; 60°C, 60 s; 95°C, 15 s.

1.3.4 Western Blot for Protein Expression

Total protein was extracted using RIPA buffer, quantified by BCA assay, and 40 μg protein was loaded for gel electrophoresis. PVDF membrane transfer conditions: LC3-Ⅱ at 300 mA for 25 min; Beclin-1 at 300 mA for 75 min; GAPDH at 300 mA for 45 min. Membranes were washed with TBST, blocked with fish gelatin, incubated with primary antibody overnight at 4°C, secondary antibody at room temperature for 2 h, and visualized using an automated chemiluminescence imaging system.

1.3.5 Immunofluorescence Detection of LC3-Ⅱ

Fixed liquid-based thin-layer slides were dried, permeabilized with autofluorescence quencher A solution in a dark box, washed, and dried. After 5% BSA blocking, slides were incubated with LC3-Ⅱ antibody (1:200) overnight at 4°C, followed by FITC Goat Anti-Rabbit IgG secondary antibody at room temperature for 2 h. Autofluorescence quencher B solution was applied, slides were washed, dried, mounted, and examined using a Zeiss imager M2 microscope with image analysis performed using lsis software (V5.8.11).

1.4 Statistical Analysis

Data were analyzed using SPSS 26.0 and GraphPad Prism 9. Normally distributed data are expressed as mean±SD and compared using paired t-tests. Non-normally distributed data are expressed as median (P25, P75) and compared using paired rank-sum tests. P<0.05 was considered statistically significant. PCR data were normalized to β-actin using the 2^(-ΔΔCt) method. Western blot bands were quantified using ImageJ.

Results

2.1 GEPIA2 Analysis of TCGA Database

Analysis of lung adenocarcinoma (483 cases vs. 347 normal) and lung squamous cell carcinoma (486 cases vs. 338 normal) revealed significantly higher Beclin-1 mRNA expression in both cancer types compared to normal tissue (P<0.05). Conversely, LC3 mRNA expression was significantly lower in both cancer types (P<0.05). The Beclin-1 result contradicted most experimental findings (Figure 4 [FIGURE:4]), prompting further experimental validation.

2.2 Real-Time PCR Results

Using β-actin as internal control, Beclin-1 mRNA expression was higher in benign pleural effusion [0.79 (0.59, 2.86)] than in malignant effusion [0.61 (0.21, 1.34)]. LC3-Ⅱ mRNA expression was also higher in benign effusion [1.94 (0.35, 2.48)] compared to malignant effusion [0.88 (0.21, 1.49)]. Both differences were statistically significant (P<0.05) (Figure 5 [FIGURE:5]).

2.3 Western Blot Results

Using GAPDH as internal control, Beclin-1 protein expression was higher in the benign group (0.936±0.335) than the malignant group (0.683±0.442). LC3-Ⅱ protein expression was also higher in the benign group (0.796±0.410) than the malignant group (0.4923±0.358). Both differences were statistically significant (P<0.05) (Figure 6 [FIGURE:6]).

2.4 Immunofluorescence

In the absence of autophagy, LC3-Ⅱ protein is diffusely distributed in the cytoplasm. During autophagy induction, LC3-Ⅱ forms green punctate or patchy fluorescent signals representing autophagosomes, with signal intensity reflecting autophagic activity. Nuclei stained with DAPI appear blue. FITC-labeled LC3-Ⅱ showed increased green punctate/patchy fluorescence in benign pleural effusion compared to malignant effusion (Figure 7 [FIGURE:7]).

Discussion

Malignant pleural effusion in lung cancer patients is associated with a median survival of only 5 months. Pleural effusion cytology remains the most common clinical method for diagnosing MPE, yet its sensitivity is merely 63%. Abnormal mesothelial cells are easily confused with tumor cells due to similar morphological features, and cytological evaluation is relatively subjective. Therefore, developing a more accurate, less invasive method for pleural effusion classification is essential. Such an approach could corroborate tumor diagnosis while reducing the invasiveness and risks associated with lung puncture, the current diagnostic gold standard. Identifying novel minimally invasive markers or therapeutic targets would significantly benefit patients.

Autophagy is primarily triggered through AMP-activated protein kinase and PI3K/AKT/mTOR signaling pathways. Upon autophagy induction, LC3-Ⅱ expression increases, forming autophagosomes that fuse with lysosomes to clear damaged organelles. Autophagy exhibits dual roles in tumor metastasis: it can suppress cancer by removing carcinogenic substrates and maintaining genetic stability, yet also promote metastasis by recycling metabolic nutrients and inducing cell dormancy. Under stress, cells initially attempt autophagy-mediated repair; however, failure can lead to autophagy-mediated programmed cell death, apoptosis, and necrosis, potentially driving malignant transformation.

Beclin-1, the first discovered mammalian autophagy-related protein, plays crucial roles in cellular metabolism and directly participates in autophagy initiation. LC3-Ⅱ serves as a specific autophagy marker throughout the process. This study utilized pleural effusion specimens to investigate Beclin-1 and LC3-Ⅱ expression in different effusion types, offering advantages over traditional cytology's limitations. While current MPE research focuses primarily on the immune microenvironment with immunological and tumor markers, autophagy-related protein expression as a molecular mechanism remains underexplored.

Our detection of Beclin-1 in pleural effusion revealed significantly higher expression in benign versus malignant effusions, consistent with previous reports of low Beclin-1 expression in tumor cells. Studies demonstrate that Beclin-1 downregulation reduces autophagy activity, promoting tumor proliferation. In adenocarcinoma mouse models, self-sustaining quiescence defects accelerate tumor growth. Loss of Ambra1, a Beclin-1 stability regulator, promotes melanoma progression through increased invasion and EMT-like processes. Recent research identifies Beclin-1 as a valuable independent biomarker for lung cancer patients, functioning as a tumor suppressor gene central to autophagy regulation and representing a prospective therapeutic target.

Interestingly, our GEPIA2 analysis of TCGA data showed higher Beclin-1 expression at the transcriptome level in lung cancer versus normal tissue, contradicting most experimental results and requiring further mechanistic investigation.

LC3-Ⅱ expression was significantly lower in lung cancer-associated pleural effusion compared to benign effusion, consistent with existing tumor studies and GEPIA2 database analysis. Increased LC3-Ⅱ expression in colorectal cancer cell lines induces cell cycle arrest and autophagy, inhibiting metastasis. LC3-Ⅱ interacts with selective autophagy substrate P62, promoting its degradation and preventing P62 accumulation that drives tumor development. P62 also interacts with pro-EMT protein Twist-1 to facilitate cell migration. Sertraline and erlotinib synergistically increase LC3-Ⅱ accumulation and autolysosome formation, effectively promoting autophagy, reducing tumor growth, and prolonging survival in NSCLC.

At the gene expression level, both Beclin-1 and LC3-Ⅱ showed higher relative expression in benign versus malignant pleural effusion. This pattern may relate to increased tumor risk associated with Beclin-1 hemizygosity. Exogenous Beclin-1 transfection inhibits tumor cell growth, while LC3-Ⅱ expression negatively correlates with lung cancer progression, possibly due to reduced Beclin-1-mediated conversion of LC3-Ⅰ to LC3-Ⅱ. Protein expression trends mirrored gene expression results, likely reflecting reduced encoding mRNA levels and potential gene mutations affecting Beclin-1 and LC3-Ⅱ. Immunofluorescence confirmed higher LC3-Ⅱ protein in benign effusion. TRAF6 interacts with Beclin-1 to activate autophagy, and TRAF6 overexpression increases LC3-Ⅱ levels, induces autophagy, degrades CTNNB1, and inhibits EMT. Beclin-1 and LC3-Ⅱ synergistically regulate autophagy.

Analysis of Beclin-1 and LC3-Ⅱ expression differences between benign and lung cancer metastatic pleural effusion may facilitate differential diagnosis and serve as effective auxiliary diagnostic markers for malignant tumors. This study provides novel perspectives for targeted treatment of pleural effusion and offers new insights for tumor diagnosis and therapy, with important implications for improving patient quality of life.

Author Contributions

Yao Wenjing and Gao Jinliang conducted the experiments and implemented the research. Wang Cuifeng and Ren Meiying conceived the study and designed the protocol; Wang Cuifeng assumes overall responsibility for the manuscript. Jing Xuefen revised the final version. Fu Yuhua performed statistical analysis and prepared figures.

Conflict of Interest

This article has no conflict of interest.

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Received: 2023-10-16; Revised: 2025-05-01
Edited by: Wang Shiyue