Targeting the Human Papillomavirus E6 and E7 Oncogenes through Expression of the Bovine Papillomavirus Type 1 E2 Protein Stimulates Cellular Motility

Abstract

Expression of the high-risk human papillomavirus (HPV) E6 and E7 oncogenes is essential for the initiationHigh-risk human papillomavirus (hrHPV)-positive cervical cancer cells harbor integrated HPV genomic DNA and are uniquely dependent upon the expression of two viral oncogenes, E6 and E7, for the maintenance of the transformed phenotype (40). hrHPV E6 is best known for its ability to target p53 for proteasomal degradation via its association with the E6-associated protein (E6-AP), a ubiquitin ligase (52,53,64). Additionally, hrHPV E6 activates telomerase to extend the life span of primary human keratinocytes (28,33,58) and binds to and deregulates several PDZ proteins that are known to regulate cell polarity, adhesion, and proliferation (27,32,41,57). This results in deregulation of tumor-suppressive activity and, in so doing, contributes to carcinogenesis. hrHPV E7 binds to and degrades members of the retinoblastoma (Rb) family, resulting in the transcription of E2F target genes such as cyclin E and cyclin A which are responsible for S-phase progression (11, 37). Additional E7 activities include the inhibition of the cyclin-dependent kinase inhibitors p21 CIP1 and p27 KIP1 which activate Rb (22, 24). Together, both hrHPV E6 and E7 contribute to carcinogenesis by suppressing apoptosis and senescence and by stimulating cellular proliferation. Consequently, a number of reports have pioneered various approaches to inhibit E6 and E7 function in HPV-positive cancer cells for the specific induction of cellular growth arrest and death (1,18,48,54). This includes the use of oncogene-specific peptide aptamers (3, 43), antisense technology (59), RNA interference (RNAi) (4,30,45), and the expression of viral E2 protein (7,39,44,56,63). Furthermore, in vivo experiments have provided proof of concept for the therapeutic targeting of E6/E7 in HPV-driven tumors in the presence and absence of conventional treatments (18,25,60).Several laboratories, including ours, have previously described the consequences of virally delivered E2 expression in HPV-positive cervical cancer cells: E6/E7 transcription ceased, followed by the reactivation of Rb and p53 tumor suppressors, cell cycle arrest, and eventually the induction of a synchronized cellular senescence phenotype. This was observed following the expression of bovine papillomavirus type 1 (BPV1) E2 transactivator (E2-TA), but not the transcriptional repressor (E2-TR), in HPV-positive cancer cells (10,17). Similar phenotypes were observed using a temperature-sensitive BPV1 E2 (E2ts) protein that is functional at the permissive temperature of 32°C but not at 37°C and above (8,62). Senescence induction in cervical cancer cells was identified by the typical flat-cell morphology (34), upregulation of senescence markers including senescence-associated ␤-galactosidase (SA-␤-Gal) activity (9), and a transcriptional profile that is reminiscent of replicatively senescent primary cells (23). Rescue of E2-expressing HeLa cells from either E6 or E7 repression further revealed that senescence...

Regulation of human genome expression and RNA splicing by human papillomavirus 16 E2 protein

“…It is of note that the alteration in the expression and splicing of genes involved in cell movement and motility were detected and that the E2 expressing cells had a reduced cellular motility when compared with wild type cells. A recent paper demonstrated that E2 can up regulate cell motility but this was done in the background of HPV positive cells making it difficult to compare with the results presented here (Morrison et al, 2011). In cervical cancers the HPV genome is integrated in the majority of cases, resulting in loss of the E2 gene (Mair et al, 2014; zur Hausen, 2009).…”

“…There have been few studies investigating the direct role of E2 in regulating host gene expression outside of the HPV cell line systems where over expression results in repression of E6 and E7 expression and reactivation of the p53 and pRb tumor suppressor pathways (Desaintes et al, 1997; Goodwin et al, 1998; Hwang et al, 1996; Johung et al, 2007; Morrison et al, 2011; Naeger et al, 1999; Parish et al, 2006; Thierry et al, 2004; Wu et al, 2000). Such reactivation results in cell cycle arrest and apoptosis and expression of E2 in these cells results in activation of genes and pathways involved in these processes.…”

“…Repression of the viral oncogenes reactivates the tumor suppressor pathways resulting in cell growth inhibition and cell death (Hwang et al, 1996; Wu et al, 2000). In some of these studies micro-array experiments have been carried out to investigate the cellular genes that are regulated by the over expression of E2 (Johung et al, 2007; Morrison et al, 2011; Naeger et al, 1999; Ramirez-Salazar et al, 2011; Thierry et al, 2004). By definition all of these studies have been carried out transiently as the cells are destined for growth inhibition or cell death, and the E2 protein is delivered by viruses or plasmid over expression that will potentially add additional toxicity to the cell.…”

Estrogen attenuates the growth of human papillomavirus positive epithelial cells

“…Downregulation of E6 and E7 expression by estrogen could result in the elevation of p53 and pRb expression (their respective tumor suppressor targets)(28–41). Previously, studies have shown that when E2 is overexpressed in HPV positive cervical cancer cells it represses transcription from the viral LCR and this repression reduces E6 and E7 levels and reactivates the p53 and pRb tumor suppressor proteins(31, 42–44, 44–50). Moreover, E2 overexpression and loss of E6/E7, results in the elevation of p53 and pRb that allows for previously observed attenuation of growth in HeLa cells(23, 31, 45, 46, 48).…”

Bovine papillomavirus productive infection in cell cultures: First evidences

“…Pancreatic keratinocyte HPV-16 E6/E7 promotes cell immortalization [70] Ectocervix keratinocyte HPV-16 E6/E7 promote cell immortalization [71] Normal foreskin keratinocyte E6 increases telomerase activity [72,73] COS7 cells HPV-1 and 8 E6 interacts with E6AP (ubiquitin) and p53 [74] Normal foreskin keratinocyte HPV-16 E6 promotes epithelium stratification reduction [75] Immortalized keratinocyte Evidences of HPV-16 productive infection [76] Murine C127 cells HPV-16 and BPV-1 E6 promotes focal adhesion decrease [77][78][79] Wart and squamous carcinoma HPV E6 inhibits apoptosis in response to UV damage [80] Normal foreskin keratinocyte HPV-16 E6/E7 induce cytogenetic aberration [81,82] Chondrocyte HPV-16 E6/E7 promotes immortalization and collagen type II expression [83] Normal foreskin keratinocyte HPV-16 E6 induces p53 degradation and p53 and hTERT expression, increasing telomerase activity [84] Normal foreskin keratinocyte HPV-16 E6/E7 induce irreversible epitelialmesenchymal transition [85] Cervical keratinocyte HPV-16 E5 interacts with endoplasmic reticulum membrane [86] Normal foreskin keratinocyte HPV-16 E6/E7 promote epigenetic alterations in host cell [87] Normal foreskin keratinocyte HPV-16 E1^E4 induce keratin reorganization [88] Normal foreskin keratinocyte BPV-1 E2 stimulates cell migration [89] Keratinocyte (PM1) HPV-8 E2, E6 e E7 promote the stem-cell phenotype acquisition [90] HEK293 cells Domain PDZ da E6 induces PKAand AKT phosphorylation [91] U2OS cells E2 binds to Brd4 regulating DNA replication [92] freezing medium (70% DMEM, 10% dymethylsulphoxyde and 20% FBS) and stocked at -196ºC. These cell lines are part of biological collection of Genetics Laboratory.…”