FANCD2 knockdown suppresses malignant phenotypes and is associated with caspase/PARP-mediated apoptosis in NCI-H2052 pleural mesothelioma cells

Our plain-English summary of FANCD2 knockdown suppresses malignant phenotypes and is associated with caspase/PARP-mediated apoptosis in NCI-H2052 pleural mesothelioma cells, by Y. Xiang, P. Su, R. Zhang, et al (Frontiers in Genetics, 2026).

The study

• Authors: Y. Xiang, P. Su, R. Zhang, D. Zhao, Z. Li, W. Yang, C. Lei, H. Chen, J. Zhang, W. Xiong
• Journal: Frontiers in Genetics
• Published: 2026;17:1926600
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Malignant pleural mesothelioma (PM) is a highly aggressive thoracic malignancy with an extremely poor prognosis and limited effective therapeutic strategies, representing a critical unmet clinical need. Fanconi anemia complementation group D2 (FANCD2) is a critical effector of the cellular DNA damage response system and plays a central role in maintaining genomic integrity. While the genoprotective functions of FANCD2 have been extensively characterized, its specific biological roles and molecular regulatory mechanisms in the malignant progression of PM remain poorly understood. The current study aimed to evaluate FANCD2 expression and its clinical associations in PM and to examine the effects of transient FANCD2 knockdown in NCI-H2052 cells.

Methods: Comprehensive bioinformatics analyses were performed based on public transcriptomic data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases to profile FANCD2 expression patterns and evaluate its clinical prognostic value in PM. Differential expression analysis was applied to confirm the aberrant upregulation of FANCD2 in PM tissues, and the associations of FANCD2 expression levels with tumor pathological staging and patient clinical prognosis were systematically assessed. Western blotting was further performed to evaluate the protein expression status of FANCD2 in clinical PM specimens and NCI-H2052 cells, with normal tissues and corresponding normal cells included as control groups. Transient FANCD2 knockdown was achieved in NCI-H2052 cells using two independent small interfering RNAs (siRNAs). A series of functional experiments were conducted to determine the effects of FANCD2 silencing on the proliferation, wound closure, and apoptosis of NCI-H2052 cells. For mechanistic exploration, the expression levels of core apoptotic proteins were detected to evaluate the association between FANCD2 depletion and Caspase/PARP-related apoptotic signaling. In addition, tumor immune infiltration analysis was performed to investigate the correlation between FANCD2 expression and M2 macrophage infiltration within the PM tumor microenvironment (TME). FANCD2 rescue and Z-VAD-FMK inhibition experiments were performed to further evaluate the involvement of caspase-dependent apoptosis.

Results: Bioinformatics results demonstrated that FANCD2 was significantly upregulated in PM tissues relative to normal control samples. Higher FANCD2 expression was associated with tumor T stage and unfavorable survival outcomes in the exploratory TCGA-MESO analysis. Consistently, Western blotting confirmed markedly increased FANCD2 protein levels in both clinical PM tissues and NCI-H2052 cells. Functional assays in NCI-H2052 cells revealed that siRNA-mediated FANCD2 knockdown significantly suppressed cell proliferation and reduced wound-closure capacity of NCI-H2052 cells, whereas it significantly increased apoptosis. Mechanistic investigations further found that FANCD2 silencing was associated with increased levels of caspase/PARP-related apoptotic markers, as evidenced by the markedly enhanced protein levels of cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, and cleaved PARP. FANCD2 restoration and pharmacological caspase inhibition partially reversed FANCD2 knockdown-induced growth suppression and apoptosis, further supporting the involvement of caspase-dependent apoptotic signaling. Moreover, TME immune correlation analysis indicated that high FANCD2 expression was positively associated with M2 macrophage infiltration in PM tumors. These findings represent exploratory correlations derived from computational immune-cell estimation.

Conclusion: In summary, FANCD2 may contribute to PM progression by supporting malignant phenotypes and limiting apoptosis. FANCD2 restoration and caspase inhibition partially reversed the growth suppression and apoptosis induced by FANCD2 knockdown, supporting the involvement of caspase-dependent apoptotic signaling. In addition, FANCD2 expression was positively associated with the computationally estimated proportion of M2 macrophages, suggesting that FANCD2 may represent an exploratory prognostic marker and a candidate therapeutic target for further investigation in PM.

Copyright © 2026 Xiang, Su, Zhang, Zhao, Li, Yang, Lei, Chen, Zhang and Xiong.

In plain English

This laboratory study examined a DNA-repair gene called FANCD2 in pleural mesothelioma. It did not measure asbestos exposure. The authors analyzed public gene databases, including 86 mesothelioma samples from the TCGA database compared with 108 non-tumor lung samples. They also tested tumor and nearby non-tumor tissue from ten surgical patients in Kunming, China, and cultured cells. FANCD2 levels were higher in tumor samples than in comparison tissue. In the database patients, higher FANCD2 was associated with poorer overall survival (log-rank P = 0.00015). The authors called this survival finding exploratory. When FANCD2 was reduced in NCI-H2052 mesothelioma cells, the cells were less viable, formed fewer colonies and closed scratch wounds less. The share of cells measured as apoptotic (early or late) was about 4.2% in control cells, against 19.5% and 24.5% with the two FANCD2-reducing treatments. Restoring FANCD2 or blocking caspase enzymes partly reversed the reduced viability, reduced colony formation and increased apoptosis. The authors concluded that FANCD2 may contribute to mesothelioma progression. They noted the database lacked complete asbestos-exposure and occupational information, and that the knockdown and rescue experiments used one cell line.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory study combining analysis of public gene-expression databases (TCGA, GEO), testing of patient tissue samples, and cell-culture experiments in which FANCD2 was reduced with small interfering RNAs (siRNAs)
• Who was studied: Database: 86 pleural mesothelioma samples from TCGA-MESO compared with 108 non-tumor lung samples, plus three GEO datasets. Clinical: tumor and paired adjacent non-tumor tissue from ten surgical patients (six male, four female; mean age 57, range 45–65) at the First Affiliated Hospital of Kunming Medical University, China, September 2022 to October 2024. Cells: expression was measured in the normal pleural mesothelial cell line MeT-5A and four mesothelioma cell lines (MSTO-211H, NCI-H28, NCI-H2052, NCI-H2452). Knockdown and rescue experiments were done in NCI-H2052 cells only.
• Exposure: No asbestos or fiber exposure was studied. The factor studied was FANCD2 gene expression. Experimentally, FANCD2 was temporarily reduced in NCI-H2052 cells using two different siRNAs. Rescue experiments either restored FANCD2 or used the caspase inhibitor Z-VAD-FMK.
• Main results:
  • FANCD2 mRNA was higher in the 86 TCGA pleural mesothelioma samples than in the 108 normal lung tissue controls (P < 0.001).
  • In the TCGA-MESO cohort, the FANCD2 expression group was significantly associated with age and tumor T stage (P < 0.05). It was not significantly associated with gender, AJCC stage, N stage, M stage or histological subtype.
  • In the TCGA-MESO cohort, high FANCD2 expression was associated with poorer overall survival (log-rank P = 0.00015) and poorer disease-free survival (log-rank P = 0.028). The overall survival analysis included 86 patients, of whom 73 died during follow-up.
  • In univariate Cox analysis, the text gives a hazard ratio for death of 2.83 (95% CI 1.72–4.66, P < 0.001) for high versus low FANCD2 expression. Table 2 prints 2.84 (1.73–4.66).
  • After adjustment for age group, sex, AJCC stage and histological subtype, high FANCD2 expression remained associated with poorer overall survival. The text gives HR = 3.02 (95% CI 1.76–5.16, P < 0.001); Table 2 prints 3.10 (1.83–5.27).
  • FANCD2 mRNA was higher in the ten patients' tumor tissues than in their paired comparison tissues (P < 0.001). FANCD2 protein was also higher in the tumor tissues.
  • FANCD2 mRNA was higher in all four mesothelioma cell lines than in the normal mesothelial cell line MeT-5A. The highest level was in NCI-H2052 cells.
  • In NCI-H2052 cells, reducing FANCD2 lowered cell viability compared with control cells at 48 and 96 hours (P < 0.01).
  • In NCI-H2052 cells, reducing FANCD2 reduced wound closure at 48 hours compared with control cells (P < 0.01). The authors stated this may reflect effects on growth and survival, not only on cell movement.
  • In NCI-H2052 cells, reducing FANCD2 caused cells to stall at an early phase of the cell cycle (G1/S arrest). It also lowered the cell-cycle proteins CDK6, CDK4 and Cyclin D1.
  • In NCI-H2052 cells, the proportion of cells measured as apoptotic (early plus late apoptosis) was about 4.2% in the control (si-NC) group. It was 19.5% in the si-FANCD2-1 group and 24.5% in the si-FANCD2-2 group.
  • In NCI-H2052 cells, reducing FANCD2 increased levels of the apoptosis markers cleaved caspase-3, cleaved caspase-8, cleaved caspase-9 and cleaved PARP. The authors stated this does not establish that FANCD2 directly regulates these proteins.
  • In NCI-H2052 cells, restoring FANCD2 partially reversed the reduced viability and colony formation caused by FANCD2 knockdown. It also lessened the increase in apoptosis.
  • In NCI-H2052 cells, the caspase inhibitor Z-VAD-FMK also partially reversed these effects of FANCD2 knockdown.
  • In TCGA mesothelioma tumors, FANCD2 expression correlated positively with computationally estimated levels of M2 macrophages. It correlated negatively with activated NK cells and activated dendritic cells. The authors treated these associations as exploratory.
• The authors' conclusion: The authors concluded that FANCD2 may contribute to pleural mesothelioma progression by supporting malignant cell behavior and limiting apoptosis. They described it as an exploratory prognostic marker and a candidate therapeutic target for further investigation.
• Limitations the authors state:
  • TCGA-MESO has no matched normal pleural tissue, so non-tumor lung samples were used for comparison. This may introduce tissue-of-origin bias.
  • The TCGA-MESO data did not provide complete information on asbestos exposure, occupational history, smoking, performance status, treatment, comorbidities or key molecular alterations. These factors could therefore not be fully adjusted for.
  • The survival analysis was exploratory. It was limited by the small TCGA-MESO cohort and the lack of an independent validation cohort.
  • The clinical tissue cohort was only ten paired specimens from one institution, which the authors said is insufficient to establish diagnostic or prognostic usefulness.
  • Cell growth was not controlled during the wound-healing assay, so reduced wound closure cannot be attributed specifically to impaired cell migration.
  • The functional experiments used only NCI-H2052 cells. The authors said the findings should not be generalized to all subtypes of pleural mesothelioma without testing other cell lines.
  • Immune-cell levels were estimated computationally and were not adjusted for tumor purity, so the immune associations are exploratory.
  • DNA-damage accumulation and replication-fork stability were not measured directly, so no specific DNA-repair mechanism was shown.
  • The BAP1 status of the NCI-H2052 cells used was not independently verified.

Topics

Medical terms in this study, explained in our Lexicon: Apoptosis, Pleural Mesothelioma, Prognosis, Staging, Macrophage, Tumor Microenvironment.

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