The Expression of HPV-16 E5 Oncoprotein Impacts the Transcript Profiles of FGFR2 and EMT-Related Genes in Preneoplastic Anal Epithelium Lesions

Abstract

Anal Squamous Cell Carcinoma (SCCA) is a rare Human Papillomavirus type 16 (HPV16)-associated carcinoma whose pathogenesis is still poorly understood. Recent studies based on biopsy and Next Generation Sequencing (NGS) approaches have linked the viral episomal status to aggressive SCCA phenotypes, suggesting a potential role of the 16E5 oncoprotein in tumor development. Our previous findings indicated that 16E5 induces Fibroblast Growth Factor Receptor 2 (FGFR2) isoform switching, aberrant mesenchymal FGFR2c expression, Epithelial Mesenchymal Transition (EMT), and cell invasion in various in vitro human keratinocyte models, as well as in the in vivo context of cervical Low-grade Squamous Intraepithelial Lesions (LSILs). To further explore the role of 16E5 in epithelial carcinogenesis, this study aims to investigate the molecular profile in HPV-related anal lesions. The results showed a significant positive correlation between 16E5 and FGFR2c, as well as 16E5 or FGFR2c and key EMT-related transcription factors, particularly in the group of HPV16 positive anal samples not containing without high grade lesions. Additionally, by coupling the molecular analysis with an interactome investigation, we hypothesized a potential functional interplay between the Ca²⁺ channel Transient Receptor Potential Ankyrin 1 (TRPA1) and FGFR2c, mediated by 16E5 during the establishment of the oncogenic signaling. These findings will help to elucidate the actual relevance of 16E5 in the early progression of anal lesions and contribute to determine its potential as target for future preventive approaches for HPV16-positive SCCA.

Keywords: E5; FGFR2c; HPV16; TRPA1; epithelial–mesenchymal transition.

Figure 1

(A) Expression levels of HPV16E5 and FGFR2 isoforms in all samples placed in ascending order of expression of 16E5; the 16E5 and FGFR2c mRNA levels were normalized with respect to W12p6 cells or HFs cells (mean values ± SD). (B) Relationships between HPV16E5 and FGFR2c expression levels (R²: Pearson’s correlation coefficient value; the regression line ±95% confidence limits and ±95% prediction bands are presented in blue and in red, respectively).

Figure 2

(A,C) Expression levels of HPV16E5 and FGFR2 isoforms in groups A and B (see Section 4) placed in ascending order of expression of 16E5; the 16E5 and FGFR2c mRNA levels were normalized with respect to W12p6 cells or HFs cells (mean values ± SD). (B,D) Relationships between HPV16E5 and FGFR2c expression levels (R²: Pearson’s correlation coefficient value; the regression line ±95% confidence limits and ±95% prediction bands are presented in blue and in red, respectively).

Figure 3

(A) Expression levels of EMT-related transcription factors Snail1, Snail2, and ZEB1 in all samples (mean values ± SD). (B) Relationships between EMT-related transcription factors regarding HPV16E5 and FGFR2c expression levels (R²: Pearson’s correlation coefficient value; the regression line ±95% confidence limits and ±95% prediction bands are presented in blue and red, respectively).

Figure 4

(A) Expression levels of EMT-related transcription factors Snail1, Snail2, and ZEB1 in group A samples. (B) Relationships between EMT-related transcription factors regarding HPV16E5 and FGFR2c expression levels (R²: Pearson’s correlation coefficient value; the regression line ±95% confidence limits and ±95% prediction bands are presented in blue and red, respectively).

Figure 5

(A) Expression levels of EMT-related transcription factors Snail1, Snail2, and ZEB1 in group B samples (mean values ± SD). (B) Relationships between EMT-related transcription factors regarding HPV16E5 and FGFR2c expression levels (R²: Pearson’s correlation coefficient value; the regression line ±95% confidence limits and ±95% prediction bands are presented in blue and in red, respectively).

Figure 6

Expression levels of TRPA1 in all samples (A) and in groups A (C) or B (E); mean values ± SD). (B,D,F) Relationships between TRPA1 and the HPV16E5 and FGFR2c expression levels (R²: Pearson’s correlation coefficient value; the regression line ±95% confidence limits and ±95% prediction bands are presented in blue and in red, respectively).

Figure 7

The BioGRID database of proteins that interact with 16E5. The Network module shows 153 proteins interaction, and among these highlighted in blue and circled in red, we find TRPA1, with which a physical interaction is indicated.