Cervical cancer (CC) is a leading cause of cancer-related deaths in women in low- and middle-income countries. Persistent infection with high-risk strains of the Human Papillomavirus (HPV) is the causative agent of CC and its oncoproteins, E6/E7, cooperate with host factors to induce and maintain CC. Therefore, identifying these host factors may have important therapeutic benefits. We have previously reported that the oncogenic transcription factor TBX3 is upregulated in CC tissues and cell lines and that it is a novel host protein that E6/E7 co-operate with to promote CC cell proliferation and migration. The present study explores the mechanism(s) that upregulates TBX3 in CC and show that c-Myc/E6/E7 transcriptionally activates TBX3. We further demonstrate that the commercial anthelmintic pyrvinium pamoate (PP) inhibits all components of this axis and inhibits several CC phenotypes.

The impact of depleting or overexpressing c-Myc, E6 and E7 by transfection with siRNA or expression constructs respectively were determined on TBX3 mRNA and protein levels by qRT-PCR and western blotting. Chromatin immunoprecipitation and luciferase reporter assays were performed to determine if c-Myc/E6/E7 transcriptionally activates TBX3 through a conserved E-box(es) in its promoter. Rescue experiments were performed and analysed in cell proliferation and scratch motility assays. CC cells were treated with PP and the impact on endogenous c-Myc/E6/E7/TBX3 was assessed by western blotting and immunofluorescence. The effect of PP was assessed on cell survival (MTT and clonogenic assays), senescence (SA-β-gal stain and p16 levels), cell cycle profile (flow cytometry), migration (scratch motility assays), and spheroid growth and invasion (calcein-AM staining and collagen matrix assays).

This study revealed that E6/E7 cooperate with c-Myc to transcriptionally activate TBX3 in CC and that this regulation required c-Myc and two E-boxes at -701 and -1210 in the TBX3 promoter. TBX3 was further shown to mediate c-Myc-induced CC proliferation and migration. These findings expose a key mechanism by with TBX3 is upregulated in CC and identifies E6/E7/c-Myc/TBX3 as a key oncogenic axis that can be targeted to treat CC. We further show that in CC, PP inhibits this axis and selectively inhibits short- and long-term cell survival, cell migration, and 3D CC spheroid formation, proliferation, and invasion.

The E6/E7/c-Myc/TBX3 axis contributes to the molecular mechanisms underpinning CC and the commercial drug PP can potentially be repurposed to target this novel oncogenic axis to treat this neoplasm and consequently reduce the burden of this disease in low- and middle-income countries.

This study was funded by the University of Cape Town, International Centre for Genetic Engineering and Biotechnology, Poliomyelitis Research Foundation, South African Medical Research Council and National Research Foundation.