Transcriptomic profile of the immune genes, oncogenes, and tumor suppressor genes in HPV associated Cervical Intraepithelial Neoplasia 3 (CIN 3) and Cervical Squamous Cell Carcinoma (CSCC): Comparable expressions indicative of invasive potential

Abstract

Cervical cancer is the fourth most common cancer among women globally, with a woman dying every 2 min. Despite the need to understand the tumor microenvironment (TME) transcriptome of cervical squamous cell carcinoma (CSCC) and cervical intraepithelial neoplasia grade 3 (CIN 3), studies remain limited. This study compares the TME transcriptome of HPV-positive CSCC and CIN 3, analyzing 168 genes involved in tumor cell interactions with inflammatory and immune mediators, transcription, signal transduction, oncogenesis, tumor suppression, angiogenesis, and apoptosis. Co-expressed genes identified in HPV + CSCC and CIN 3 were analyzed using computational biology. Gene Ontology and KEGG enrichment identified relevant biological pathways and cancer hallmarks. Fifty-five co-expressed genes were linked to cancer pathways, inflammatory responses, cell migration, and development. KEGG enrichment highlighted viral protein interactions involving cytokines, IL-17 signaling, and chemokine receptor interactions. These genes were associated with cancer hallmark pathways, including angiogenesis, inflammation, proliferation, genomic instability, invasion, and metastasis. Their similar expression in CSCC and CIN 3 suggests a potential prognostic value and that CIN 3 progression may involve changes in gene expression. We propose the term "CSCC-like carcinoma," indicating CIN 3's increased invasive potential at the molecular level.

Keywords: Cervical squamous cell carcinoma; High-grade cervical intraepithelial neoplasia; Human papillomavirus; Tumor microenvironment.

Graphical abstract

Fig. 1

Comparison of Fold Changes in Tumor Inflammation and Immunity Crosstalk Gene Expression Between CIN 3 and CSCC Patients. (A, B, C, and D) showed the fold changes in the expression of oncogenes and tumor suppressor genes between CIN 3 and CSCC patients. The height of the blue bars (CIN 3) and red bars (CSCC) for each gene indicates the mean fold change in expression. (E) Voronoi treemap illustrates the hierarchical relationships of gene expression. The area of each convex polygon corresponds to the fold change in gene expression for each gene in CSCC and CIN 3 patients, visually representing their relative fold differences.

Fig. 2

Comparison of Fold Changes in Oncogenes and Tumor Suppressor Gene Expression Between CIN 3 and CSCC Patients. (A, B, and C) The fold changes in the expression of oncogenes and tumor suppressor genes between CIN 3 and CSCC patients. The height of the blue bars (CIN 3) and red bars (CSCC) for each gene indicates the mean fold change in expression. (∗ = P < 0.05, ∗∗ = P < 0.01 and ∗∗∗ = P < 0.001). (D) Voronoi Treemaps Representing Gene Expression Hierarchies. The area of each convex polygon corresponds to the fold change in gene expression for each gene in CSCC and CIN 3 patients, representing their relative differences. The color scale on the right side represents fold changes in the expression levels.

Fig. 3

Downregulation of Tumor Inflammation and Immunity Crosstalk Genes in CIN 3 and CSCC Patients. The relative positioning of the blue and red data points for each gene indicates the direction and magnitude of the fold change. Blue data points represent CIN 3 and red data points represent the CSCC.

Fig. 4

Comparison of downregulation of the fold changes in oncogenes and tumor suppressor gene expression between CIN 3 and CSCC patients. The relative positioning of the blue and red data points for each gene indicates the direction and magnitude of the fold change. Blue data points represent CIN 3 and red data points represent the CSCC.

Fig. 5

The ring heatmap clustergram of oncogene and tumor suppressor gene expression in a subset of CIN 3 (n = 16) and CSCC (n = 34) FFPE samples. The heatmap color scale indicates the range of expression values the color gradient represents from low (blue) to high (red) expression. Genes with similar colors across samples were co-expressed. The dendrogram (tree structure) showed the hierarchical clustering of genes based on their expression patterns. Due to sample volume constraints, only these 50 samples were included in the visual representation.

Fig. 6

The ring heatmap clustergram of oncogene and tumor suppressor gene expression in a subset of CIN 3 (n = 11) and CSCC (n = 20) FFPE samples. Rings represent different HPV-associated CIN 3 and CSCC. The heatmap color scale indicates the range of expression values the color gradient represents from low (blue) to high (red) expression. Genes with similar colors across samples were co-expressed. The dendrogram (tree structure) showed the hierarchical clustering of genes based on their expression patterns. Due to sample volume constraints, only these 31 samples were included in the visual representation.

Fig. 7

Heatmap Correlation of Various Immune-Related Genes in CSCC and CIN 3. (A) Correlation analysis of gene expression in CSCC patients. (B) Correlation analysis of gene expression in CIN 3 patients. Blue color indicates negative correlations and red color indicates positive correlations. The intensity of the color corresponds to the magnitude of the correlation coefficient.

Fig. 8

Heatmap Correlation of Various Oncogenes and tumor suppressor genes in CSCC and CIN 3. (A) Correlation analysis of gene expression in CSCC patients. (B) Correlation analysis of gene expression in CIN 3 patients. Blue color indicates negative correlations and red color indicates positive correlations. The intensity of the color corresponds to the magnitude of the correlation coefficient.

Fig. 9

Quantification of 12 human proinflammatory by multiplex cytokine ELISA in FFPE-derived protein lysates from CIN 3: n = 20, CSCC: n = 34, and HPV-negative control cervical tissue n = 13. Error bars represent mean ± SEM. Control samples were derived from patients undergoing hysterectomy for benign gynecological indications. Statistical analysis was performed using two-way ANOVA followed by Tukey's multiple comparisons tests. (A) IL1-α (B) IL-1β. (C) IL-2. (D) IL-4 (E) IL-6. (F) IL-8. (G) IL-10. (H) IL-12. (I) IL-17A. (J) IFN-γ. (K) TNF-α and (L) GM-C.

Fig. 10

Enrichment of co-upregulated genes from CIN 3 and CSCC across the Cancer Hallmarks pathways. Bar height represents the number of mapped genes per hallmark. The full gene list is provided in Supplementary Table.

Fig. 11

(A) Top 20 Enriched Gene Ontology Clusters with their representative enriched terms of co-expressed genes analyzed by Metascape. (B) KEGG pathway enrichment. Enriched ontology clusters were colored by cluster-ID; the nodes that shared the same cluster-ID typically clustered close to each other. The thickness of the edge represents the similarity score.