Effect of HPV E6/E7 siRNA with Chemotherapeutic Agents on the Regulation of TP53/E2F Dynamic Behavior for Cell Fate Decisions

Abstract

Toxicity and resistance remain major challenges for advanced or recurrent cervical cancer therapies, as treatment requires high doses of chemotherapeutic agents. Restoration of TP53 and hypophosphorylated-retinoblastoma (pRB) proteins by human papillomavirus (HPV) E6/E7 siRNA sensitizes HPV-positive cervical cancer cells toward chemotherapeutic agents. Here, we investigated the therapeutic effects of E6/E7 siRNA on the dynamic behavior of TP53 and RB/E2F signaling networks in deciding the cell fate. The synergistic effect of HPV E6/E7 siRNA pool (SP) with chemotherapeutic agents on TP53 and RB/E2F signaling, proliferation, and apoptosis was analyzed in vitro and in vivo. Compared to the E6/E7 SP alone, E6/E7 SP with cisplatin treatment effectively restored TP53 and RB/E2F signaling and contributes to differences in cell fate, such as apoptosis or cell cycle arrest. We also developed a cellular dynamics model that includes TP53-RB/E2F dynamics and cell proliferation profiles, and confirmed its utility for investigating E6/E7 siRNA-based combination regimens. Using a dual reporter system, we further confirmed the cross talk between TP53 and RB/E2F signaling mechanisms. Treatment of E6/E7 SP cationic liposome (i.v.) with cisplatin and paclitaxel (i.p.) potentially inhibited tumor growth in BALB/c-nude mice. Altogether, our findings suggest that stabilization of TP53 and the RB/E2F repressor complex by E6/E7 SP combined with low-dose chemotherapy can effectively suppress tumor growth.

Biological and clinical aspects of HPV-related cancers

“…Another study also reported suppressed cell growth in HPV-related cervical cancer cells after downregulating HPV 16 E6 and E7 using an adenovirus-mediated transfection which correlated with higher expression of p53 and/or pRb proteins 50 , 51 . Additionally, a similar effect of increased sensitization to cisplatin treatment has been shown in cervical cancer cells after treatment with siRNA against the HPV 16 and HPV 18 E6 and E7 52 . Furthermore, downregulation of HPV 16 and HPV 18 E6 and E7 led to radiosensitizing effects and suppressed growth in cervical cancer cells and xenograft mouse tumors 53 .…”

Dysregulation of miR-375/AEG-1 Axis by Human Papillomavirus 16/18-E6/E7 Promotes Cellular Proliferation, Migration, and Invasion in Cervical Cancer

“…Furthermore, HPV 16, 18 E6/E7 siRNA significantly inhibits invasion of CC cells (Figures 7J,K). We also confirmed the p53 restoration and Rb expression level in our previous studies by using the HPV 16/18 E6-E7 siRNA (39, 41). We hypothesized that HPV 16 and 18 E6-E7 viral oncogenes could induce differential miR-375 expression which contributed to the cervical tumorigenesis.…”

“…For siRNA transfection, AEG-1 siRNA 1 (referred as AEG-1 siRNA) (sense: 5′-GACACUGGAGAUGCUAAUAUU-3′, antisense: 5′-UAUUAGCAUCUCCAGUGUCUU-3′), AEG-1 siRNA 2 (sense: 5′-GGUGAAGAUAACUCUACUGUU-3′, antisense: 5′-CAGUAGAGUUAUCUUCACCUU-3′) and their negative control siRNA (SIC008) were synthesized and purchased from sigma aldrich, USA. A pool of three different siRNA for HPV 16 E6/E7 and HPV 18 E6/E7 (kindly gifted from Prof. Young Kee Shin, Research Institute of Pharmaceutical Science, Seoul National University, Seoul, Republic of Korea) (39), and for miRNA transfection, miR-375 mimic (HMI0537), miR-mimic-negative control (HMC0002), miR-375 inhibitor (HSTUD0537), and miR-inhibitor-negative control (NCSTUD001) (Sigma Aldrich, USA) were transiently transfected into cells at a concentration of 20 nM using Lipofectamine RNAiMAX Reagent (Invitrogen). The mock control cells were treated with transfection reagent alone and the cells were maintained for 48 h after transfection.…”

Co-Delivery of p53 Restored and E7 Targeted Nucleic Acids by Poly (Beta-Amino Ester) Complex Nanoparticles for the Treatment of HPV Related Cervical Lesions

“…We hypothesized that p53 overexpression may restore RB expression through some pathways. Previous reports have confirmed the cross-talk between p53 and Rb/E2F signaling mechanisms (Rajasekaran et al, 2017). We can see that E7 is still expressed in the basal region.…”