The mechanistic role of the thromboxane A2 receptor (TBXA2R) in non-small cell lung cancer (NSCLC)

Fig. 1. Elevated TBXA2R expression in human lung cancer and its impact on patient survival. A, immunohistochemical staining was performed to assess TBXA2R protein levels in both normal lung tissues and lung cancer specimens. TBXA2R was visualized using DAB (brown) staining, with hematoxylin used for counterstaining the nuclei (blue). Quantitative analysis of staining intensity was conducted, and statistical significance was evaluated using one-way ANOVA. The analysis included normal lung tissues (n = 10), adenocarcinoma tissues (n = 44), and squamous cell carcinoma tissues (n = 41); the scale bar denotes 100 μm. B, Kaplan-Meier survival analysis based on TBXA2R expression levels was conducted using the Kaplan-Meier Plotter tool (http://kmplot.com/analysis), incorporating Affymetrix probe IDs 207554_x_at, 207555_x_at, 211590_x_at, and 336_x_at for TBXA2R. A significant difference in TBXA2R expression between normal lung and adenocarcinoma tissues is indicated by asterisks (***, p < 0.001, one-way ANOVA)

Fig. 2. TBXA2R knockdown inhibits the proliferation of H1650 and H441 lung cancer cell lines. A, comparative analysis of TBXA2R expression levels in normal human lung cells versus multiple lung cancer cell lines. B, establishment of stable TBXA2R knockdown in H1650 lung cancer cells, confirmed by Western blot analysis. C, assessment of the effect of TBXA2R knockdown on the anchorage-independent growth of H1650 cells. D, generation of H441 lung cancer cells with stable TBXA2R knockdown. E, analysis of TBXA2R knockdown impact on the anchorage-independent growth of H441 cells. Cells stably expressing either non-targeting shRNA (shNT) or TBXA2R-targeting shRNA (shTBXA2R) were cultured in 1.25% agar, and colony formation was quantified using microscopy and Image-Pro Plus software (version 6). Results are presented as mean values ± SD from 3 independent experiments. Statistical significance was determined using the Student’s t-test, with asterisks indicating significant differences between TBXA2R knockdown and control groups. F, G, immunofluorescence analysis of 3D multicellular spheroids derived from H1650 and H441 cells. TBXA2R is indicated in red, with nuclei counterstained with DAPI…

Fig. 3. RNA-seq analysis of the gene changes in response to TBXA2R knockdown (shTBXA2R) in H1650 cells. A, comparison of TBXA2R expression levels between shTBXA2R and WT (shCon) as determined by RNA-seq data. B, volcano plot depicting gene expression changes (shTBXA2R vs. shCon; fold change ≥ 2; p value < 0.05). C, KEGG analysis of differentially expressed genes presented in a bubble chart. D, cluster plot showcasing the expression profiles of genes associated with the identified signaling pathways. E, fold changes and p values of genes in the PI3K-AKT pathway. F, fold changes and p values of genes in the cytokine-cytokine receptor interaction pathway

Fig. 4. Effects of TBXA2R knockdown on the PI3K/AKT/mTOR signaling pathway and NF-κB activation in lung cancer cells. A, TBXA2R knockdown results in decreased phosphorylation levels within the PI3K/AKT/mTOR signaling pathway. B, C, H1650 and H441 cells with TBXA2R knockdown (shTBXA2R) and control (shCon) were treated with urethane for specified durations; subsequent NF-κB nuclear translocation was evaluated using a luciferase assay. D, E, differential expression of key genes in the PI3K-AKT pathway (PCK1, LPAR4) and the cytokine-cytokine receptor interaction pathway (CXCL4, INHBC) was confirmed through RT-PCR analysis. Data are presented as means ± SD from triplicate experiments conducted independently 3 times (*, p < 0.05; **, p < 0.01, one-way ANOVA)

Fig. 5. Knocking down TBXA2R expression suppresses EGFR by inhibiting NF-κB p52 nuclear translocation in lung cancer cells. A, RNA-seq analysis comparing EGFR expression levels between TBXA2R knockdown (shTBXA2R) and wild-type (shCon) cells. B, Western blot analysis illustrating the impact of TBXA2R knockdown on EGFR protein levels in H1650 and H441 cell lines. C, ChIP-qPCR showing the interaction of the NF-κB protein with the EGFR gene promoter. D, knockdown of TBXA2R leads to a decrease in EGFR expression by modulating its promoter activity through NF-κB p52. E, predicted binding motif for NF-κB p52 within the EGFR promoter region. F, immunofluorescence analysis of H1650 cells with stable TBXA2R knockdown, treated with urethane for 24 h, assessing NF-κB p52 localization (scale bars represent 50 μm). Data are presented as means ± SD from 3 independent experiments conducted in triplicate (*, p < 0.05; **, p < 0.01, one-way ANOVA)

Fig. 6. Loss of TBXA2R inhibits urethane-induced lung carcinogenesis. A, WT and TBXA2R KO mice were used in the study. Mice received intraperitoneal injections of urethane (1 g/kg in PBS) or vehicle (PBS) once a week for 10 weeks. Lungs were collected 30 weeks following the initial urethane administration. B, body weight comparison across different groups. C, tumor multiplicity analysis revealed an average of 4.2 ± 2.1 tumors in urethane-treated TBXA2R KO mice compared to 10.1 ± 4.1 tumors in the urethane-treated WT group. No significant sex differences in tumor numbers were observed between male and female mice. D, lung tissue samples were harvested and stained with hematoxylin and eosin (H&E). Immunohistochemical analysis was performed to assess the expression of proliferating cell nuclear antigen (PCNA) in the lungs of urethane-treated mice versus vehicle-treated controls. Data are presented as means ± SD (*, p < 0.05; **, p < 0.01, one-way ANOVA)