Prognostic Risk Models Using Epithelial Cells Identify β-Sitosterol as a Potential Therapeutic Target Against Esophageal Squamous Cell Carcinoma

Figures & data

Figure 1 Analysis of single-cell sequencing data. (A) Reduced dimensional cluster analysis, t-distributed stochastic neighbor embedding (t-SNE) distribution of different cell clusters. (B) A violin diagram showing the marker genes for each cell type. (C) Distribution of t-SNE in different cell types. The red arrow indicates the reference gene of epithelial cells.

Figure 2 Analysis of epithelial cell signaling pathways. (A–C) Network diagram depicting the interaction of the Wnt, transformation growth factor (TGF), and epidermal growth factor (EGF) signaling pathways. (D–F) Heatmap of the interaction of the Wnt, TGF, and EGF signaling pathways. (G–I) AUCell analysis based on the Reactome pathway database.

Figure 3 Acquisition of differentially expressed genes. (A) Principal component analysis plot of the GSE161533 and GSE20347 dataset concatenation. (B) Volcano map of merged differentially expressed genes. (C) Uniform Manifold Approximation and Projection plot showing differentially expressed genes of different cell types. (D) Wayne diagram of differentially expressed genes and epithelial cells in ESCC.

Figure 4 Immuno-infiltration analysis. (A) ESTIMAT analysis of the high- and low-risk group. (B) CIBERSORTx analysis of the abundance of immune cells. (C) Heatmap of correlation between risk score and the presence of immune cell types. (D) The single-sample Gene Set Enrichment Analysis scores of 28 immune cells in the high- and low-risk groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Abbreviation: ns, Not Statistically Significant.

Table 1 Detailed Information on LAPTM4B, CTSL, MYO10, NCF2, and PDLIM2

Gene Symbol	Gene ID	Full Name	Function
LAPTM4B	7805	Lysosomal protein transmembrane 4 beta	Involved in vesicle trafficking, lysosome maturation and fusion, as well as apoptosis and tumor development. LAPTM4B is highly expressed in a wide range of human tumors and correlates with tumor invasion and metastasis, treatment resistance, and other characteristics.
MYO10	4646	Myosin X	MYO10 plays an important role in the process of cell movement and cell architecture. Its main functions involve directional cell movement, cell adhesion and migration.
CTSL	1514	Cathepsin L	CTSL is an important lysosomal enzyme that performs multiple functions within the cell. It is involved in intracellular protein degradation, lysosome formation and functional maintenance. CTSL plays an important regulatory role in physiological processes such as immune response, apoptosis, embryonic development, tissue remodeling and inflammation.
NCF2	4759	Neutrophil cytosolic factor 2	It is a multisubunit enzyme complex involved in electron transfer during intracellular reactions to produce reactive oxygen species. The main function of NCF2 is to provide electrons for NOX and to promote intracellular redox reactions.
PDLIM2	10,350	PDZ and LIM domain 2	PDLIM2 protein is a binding protein that interacts with other proteins and forms complexes involved in the regulation of multiple cellular processes and signaling pathways. PDLIM2 plays an important role in physiological processes such as cell development, differentiation and proliferation.

Figure 5 Screening and validation of core prognostic genes. (A) Prognostic forest map. (B) LASSO analysis to construct a fitted model. (C) Determining the best lambda value for the fitted model: the x-axis represents the logarithmized lambda value, the y-axis represents the error of the model, and the dotted line on the left represents the lambda value that minimizes the error and the number of features screened. (D–H) Dataset GSE45670 validation. (I–M) Validation of the GEPIA dataset. *p < 0.05.

Abbreviations: N, Total sample size; HR, Hazard Ratio.

Figure 6 Nomogram construction of related genes. (A) Nomogram construction. (B) ROC curves and calibration curves for the nomograms in the training group. (C) ROC curves and calibration curves for the nomograms in the validation group.

Figure 7 Epithelial cell analysis. (A) The t-distributed stochastic neighbor embedding (t-SNE) of 10 cell clusters. (B) Dot plot of different marker genes. (C) Identification of tumor and paratumor marker genes; (D) Copy number variation profile showing relative differences between the tumor and paratumor groups. (E) Violin plot of essential genes.

Table 2 Detailed Information on the Top 8 Sensitive Drugs

Drug Name	Drug Target	Target Pathway	Introduction
AZD8186	PI3Kalpha, PI3Kbeta	PI3K/MTOR signaling	AZD8186 interferes with intracellular signaling pathways involved in processes such as cell growth, differentiation, and apoptosis by inhibiting PI3K kinase.
Entospletinib	SYK	Other, kinases	Entospletinib slows the growth and proliferation of cancer cells by inhibiting signaling pathways by targeting BTK. BTK is a tyrosine kinase involved in B-cell receptor signaling and other inflammation-related pathways.
Gemcitabine	Pyrimidine antimetabolite	DNA replication	It is a nucleoside analog that inhibits the proliferation and division of cancer cells by interfering with their DNA and RNA synthesis.
GSK2606414	PERK	Metabolism	GSK2606414 interferes with the proliferation and growth of cancer cells by inhibiting the mTOR signaling pathway. mTOR is a key intracellular signaling pathway involved in cell growth, division, metabolism and survival.
Mitoxantrone	TOP2	DNA replication	The mechanism of action of Mitoxantrone mainly involves DNA cross-linking and damage. It blocks the division and proliferation of cancer cells by interacting with DNA and interfering with the stability and replication process of DNA strands.
PRT062607	SYK	Other, kinases	PRT062607 blocks signaling in these diseases by inhibiting BTK activity, reducing the proliferation and survival of pathological B cells.
SB505124	TGFBR1, ACVR1B, ACVR1C	RTK signaling	It is a selective transforming growth factor beta (TGF-β) receptor kinase (ALK4/5/7) inhibitor, which means that it interferes with the normal function of the TGF-β signaling pathway
UMI-77	MCL1	Apoptosis regulation	UMI-77 acts as a dopamine D2S receptor agonist that mimics the effects of dopamine and selectively activates D2S receptors.

Figure 8 Drug sensitivity analysis. (A–H) Top 8 drugs showing the most sensitivity with Risk score. High/Low indicates the grouping of samples into high and low categories. p represents the result of statistical analysis, with p<0.05 indicating significance.

Figure 9 Molecular docking of β-sitosterol with five core genes and the mRNA levels were measured by real-time fluorescence quantitative PCR. (A-E) Molecular docking of β-sitosterol to five core genes. (F) The docking results were presented in a heatmap. (G) K-M survival analysis of the five core genes in the TCGA database. (H) Further validation of the core genes in the external dataset GSE53624.

Figure 10 Overexpression of PDLIM2 significantly inhibits the proliferation and migration of KYSE150 and Eca9706 cells. (A) q-PCR detection of PDLIM2 expression in esophageal squamous cell carcinoma cell lines and normal esophageal epithelial cell lines. (B and C) q-PCR and WB were used to detect the overexpression efficiency of PDLIM2 in KYSE150 and Eca9706 cells. (D) The effect of PDLIM2 on the migration of KYSE150 and Eca9706 cells was explored in wound healing assays. (E) Compared with the control group, overexpressed PDLIM2 significantly inhibits the migration and invasion of esophageal squamous carcinoma cells. (F) The effect of PDLIM2 on the proliferation of KYSE150 and Eca9706 cells was investigated in colony formation assays. **p < 0.01, ***p < 0.001. Scale bars: 100 µm.

Data Sharing Statement

Data will be made available on request. For further information, please contact the corresponding author.