Inhibition of p16 and NF-κB Oncogenic Activity in Human Papillomavirus-Infected Cervical Cancer Cells: A New Role for Activating Transcription Factor-3

Abstract

Objective: Activating transcription factor 3 (ATF3) has attracted recent scientific attention as a novel mediator of tumor suppression, particularly within the context of cervical cancer (CC). Our prior research demonstrated that ATF3 overexpression induces cell cycle arrest and apoptosis in human papillomavirus (HPV)16- and HPV18-positive CC cells. The present study aims to examine the impact of ATF3 overexpression on the expression levels of p16 and NF-κB, two proteins with pro-tumorigenic roles in HPV-induced CC. Methods: Ca Ski and HeLa cells underwent transfection with pCMV6-AC-IRES-GFP plasmids containing the ATF3 gene. To establish the optimal plasmid DNA quantities for transfection, MTT assay was conducted. Furthermore, fluorescence microscopy and flow cytometric analysis were employed to assess the efficiency of transfection. The expression levels of p16 and NF-κB were evaluated by RT-qPCR and western blotting prior and subsequent to ATF3 overexpression. Results: The overexpression of ATF3 induced a decrease in p16 mRNA levels in both Ca Ski and HeLa cells (p<0.04), along with the concomitant reduction of p16 protein expression within both cellular populations (p<0.005). Additionally, it led to a reduction in NF-κB p65 protein levels in both cell lines (p<0.005), with no discernible impact on its mRNA expression. Conclusion: Given ATF3's demonstrated capability to downregulate p16 and NF-κB, both of which play important pro-tumorigenic roles in HPV-related CC, ATF3 emerges as a promising therapeutic candidate with the potential for application in the treatment of CC.

Keywords: ATF3; HPV; NF-κB; cervical cancer; p16.