Transcriptomic analysis of the effects of the HPV18 E6E7 gene on the cell death mode in esophageal squamous cell carcinoma

**Background:** Esophageal carcinoma (ESCA) is the seventh most common cancer worldwide, with squamous cell carcinoma (SCC) comprising 90% of cases. Human papillomavirus (HPV) infection is a known risk factor, and the authors' previous work demonstrated that HPV18 E6E7 induces immortalization and malignant transformation of human embryonic esophageal epithelial cells. However, the mechanisms by which HPV affects cell death pathways in ESCA remain poorly understood. This study aimed to investigate the effects of HPV18 E6E7 on regulated cell death (RCD) pathways—apoptosis, autophagy, and pyroptosis—in ESCC at the transcriptional level.

**Methods:** RNA-Seq raw data for HPV18-positive (EC9706, EC109) and HPV-negative (HKESC01) ESCC samples were obtained from NCBI BioProject (PRJNA530677). Differential expression analysis was performed using edgeR, with genes selected at |log2(Fold change)| >2 and padj <0.05. GO and KEGG analyses were conducted using OmicsBean. TCGA expression and survival analyses were performed using GEPIA2, and tumor-associated fibroblast infiltration was analyzed via TIMER2.0. For experimental validation, HPV18 E6E7 was cloned into a pLVX-puro lentiviral vector and stably transfected into KYSE150 and KYSE180 cells. Apoptosis was induced with cisplatin (20, 40, 80 µM) and detected by Annexin V FITC flow cytometry. Autophagy was induced with HBSS and assessed using LC3-RFP-GFP confocal microscopy and western blotting for LC3B. Pyroptosis was induced with 15 mM α-ketoglutarate and observed by microscopy. Gene expression was measured by RT-qPCR.

**Key Results:** Comparing HPV18-positive vs HPV-negative cells, 5,393 differentially expressed genes were identified (2,016 upregulated, 3,377 downregulated). KEGG analysis showed enrichment in PI3K/Akt signaling (P=4.47e-06), retinol metabolism (P=1.92e-05), and cytokine-cytokine receptor interaction (P=2.03e-05). Multiple RCD pathways were significantly enriched. For apoptosis, CDKN2A was highly expressed while DSG3 and PKP1 were reduced in HPV18-positive cells. HPV18 E6E7 significantly promoted CDKN2A expression and inhibited DSG3 expression. Cisplatin (80 µM) induced apoptosis in KYSE150 cells, but HPV18 E6E7 significantly inhibited this effect. For autophagy, ULK1 and ADRB2 mRNA expression was reduced in HPV18-positive cells. HPV18 E6E7 inhibited autophagosome-lysosome fusion in late autophagy without affecting LC3-I to LC3-II cleavage. For pyroptosis, GSDMA, GSDMC, NLRP3, AIM2, NLRP1, and TLR1 were all reduced in HPV18-positive cells. HPV18 E6E7 significantly inhibited GSDMC and TLR1 expression. α-ketoglutarate-induced pyroptosis was clearly observed in HPV-negative cells but was limited in HPV18 E6E7-expressing cells.

**Clinical Implications:** This study demonstrates that HPV18 E6E7 inhibits multiple cell death pathways in ESCC, which may contribute to chemoresistance and tumor progression. The finding that HPV18 E6E7 increases resistance to cisplatin-induced apoptosis has direct clinical relevance, as cisplatin is a commonly used chemotherapeutic agent for ESCC. The identification of specific genes regulated by HPV18 E6E7 (CDKN2A, DSG3, ULK1, ADRB2, GSDMC, TLR1) provides potential biomarkers and therapeutic targets. The observation that HPV inhibits pyroptosis through GSDMC suppression is particularly novel and suggests that restoring pyroptosis pathways could be a therapeutic strategy. Limitations include the use of only two HPV18-positive cell lines and the need for future studies using HPV18 E6E7 knockout models to confirm these findings.