Transcriptome analysis of HPV16-positive cells expressing intrabodies targeting E6 and E7 oncoproteins to unravel intrabody antitumor activity

Background: Human Papillomavirus-associated cancer remains a global health issue despite the availability of prophylactic vaccination. Two single-chain recombinant antibodies specific for the high-risk HPV16-E6 and E7 oncoproteins were previously developed as intrabodies in HPV16-positive cells. The anti-E6 I7NUC and anti-E7 43M2SD intrabodies demonstrated antiproliferative efficacy in vitro and in mouse tumor models by interfering with p53 and pRb tumor suppressors. This study aimed to explore the mechanism behind this antitumor activity. Methods: HPV16-positive SiHa cells were electroporated with the intrabody DNA plasmids and processed for RNA-seq analysis 6 h after transfection. Differential expression and functional analysis were conducted to identify significantly modulated genes and enriched pathways. RT-qPCR was employed to confirm the modulation of selected genes. The human interactome of E6 and E7, retrieved from BioGRID, was used to check for known interactions. Results: The anti-E6 I7Nuc modulates 12,820 genes, of which 6,072 are upregulated and 6,748 downregulated, mainly encoding proteins that directly bind to HPV16E6. The anti-E7 43M2SD intrabody modulates 1,174 genes, mostly encoding proteins that interact indirectly with E7 binders. Notably, 3,191 genes in cells expressing I7NUC and 468 in those expressing 43M2SD are novel. The functional analysis revealed over 100 KEGG pathways significantly affected by I7NUC and only one pathway affected by 43M2SD. “Neuroactive ligand-receptor interaction, Proteasome, Nucleocytoplasmic transport, Protein processing in endoplasmic reticulum, Cell cycle, ATP-dependent chromatin remodelling, Cellular senescence, Ubiquitin-mediated proteolysis, Ribosome and Viral carcinogenesis” are the top significant pathways affected by I7NUC, with almost 100% down-modulated DEGs. 43M2SD affected only the “Viral protein interaction with cytokine and cytokine receptor” pathway, with CXCL6, 9, 10 and 11 genes all downregulated. I7NUC determines the transcriptional downregulation of Energy Metabolism pathway enzymes, including SLC2A1, SLC5A1, hexokinase1/2, LDHA, GLS2, SLC7A5, SLC7A11, HIF-1, and a significant percentage of transcripts associated with the phosphorylation-oxidative enzyme complexes. Conclusions: The anti-E6 and anti-E7 intrabodies interfere with cellular pathways essential for tumor transformation, confirming their great potential as therapeutic agents for HPV16-related diseases. The differences observed in the effects of the two intrabodies at the transcriptional level may be advantageous in clinical applications to different tumor types or grades of premalignant lesions.