6.2. Histone Rearrangement
Favre and collaborators were the first to describe that the HPV genome is associated with the canonical histones H2A, H2B, H3 and H4
[13][17]. It was reported that the E2, E6 and E7 proteins of HR-HPV have the capacity not only to bind to the CBP/p300 coactivator complex and inhibit its histone acetyltransferase (HAT) activity, but also to block the ability of p300 to activate p53-responsive promoter elements. This results in the deregulation of cellular signaling, which decreases genome stability and favors the cellular transformation process
[48][49][50][51][105,106,107,108].
7. Loss of E2 Protein Function
It is generally understood that the oncogenic HPV E2 protein is a negative regulator of the expression of the E6 and E7 oncogenes
[52][72]. It is understood that the loss of E2 function can occur in two ways: the first is through the integration process, where breaks in the E1/E2 regions lead to the functional loss of the E2 gene
[52][53][54][55][72,76,109,110]; and the second involves the methylation of the CpG sites located in the E2BSs of the HPV LCR, specifically, E2BS1, E2BS3 and E2BS4, which results in the activation of the p97 promoter and the subsequent loss of the repressive function of the E2 protein on the transcription of E7/E6
[56][57][58][59][60][96,111,112,113,114]. Therefore, the loss of E2 function could be considered a key step in carcinogenesis.
8. Deregulated Expression of the E6/E7 Oncogenes
Various studies reported that the loss of E2 function—either due to the phenomenon of viral genome integration or due to the methylation of the E2BSs in the HPV LCR—is associated with the overexpression or the aberrant expression of E6 and E7
[61][62][63][64][102,115,116,117]. However, these same studies mentioned that there was no significant difference when comparing the expression levels of the E6/E7 oncogenes in samples that expressed E2 and contained HPV genomes in a purely episomal state or in a coexisting state, with those in samples that contained HPV genomes in a purely integrated state, without E2 expression. This indicates that methylation at specific sites of the E2BSs in the LCR plays an important role not only in the loss of E2 function in those samples harboring transcriptionally active E2 genes, but also in regulating the expression level of E6/E7. This suggests that the overexpression of the E6/E7 oncogenes can be favored only in cases where the following criteria are met: (1) there is a high number of viral genomes in the episomal state with intact E2 genes and with site-specific methylation in E2BS-I and -II; (2) there is a low or moderate viral load, and the viral genomes are integrated at distal sites in a single copy and probably under the control of strong promoter regions in the host cell genome
[61][64][102,117].
9. Regulation of Host Cell Protein Function
It was reported that the E6 oncoprotein of HR-HPV can evade cell death by apoptosis through two pathways. The first is through the proteasomal degradation of p53 via its association with the ubiquitin ligase UBE3a (E6AP)
[65][66][67][118,119,120]. The second is through the interaction of E6 with hADA3—a protein that functions as a coactivator of p53-mediated transactivation for a variety of target promoters—where E6 induces the degradation of hADA3, thus inactivating the function of p53 and overriding the arrest of p14ARF-induced cell growth, despite the presence of normal levels of p53
[68][69][121,122]. Conversely, it is widely accepted that the E7 oncoprotein of HR-HPVs plays two main roles to induce the transforming and proliferative process in cells. Firstly, it binds with members of the retinoblastoma protein (pRb) family, such as p107 and p130
[70][123], which promotes the transcriptional activity of E2F transcription factors, thus regulating cell cycle entry and the progression from the G1 phase to the S phase of the cell cycle
[71][124]. Secondly, it destabilizes pRb via degradation through the ubiquitin–proteasome pathway, leading to oncogenic transformation
[72][125].
10. Acquisition of the Mesenchymal Phenotype
An established feature of solid tumors which are not associated with oncogenic viruses is the acquisition of a mesenchymal phenotype. This is characterized by the overexpression of N-cadherin, vimentin, fibronectin, Twist, FOX C2, SOX 10, MMP-2, MMP-3, MMP-9, Snail and Slug (currently designated as Snai1 and Snai2, respectively, by the HUGO Gene Nomenclature Committee) and a decrease in the expression of E-cadherin (currently designated as CDH1 by the HUGO Gene Nomenclature Committee), desmoplakin, cytokeratin and occludin
[73][74][203,204]. Nevertheless, Hellner and collaborators
[75][205] reported that both E6 and E7 induced the expression of N-cadherin and that the expression of E7 in primary human foreskin keratinocytes (HFK) induced elevated levels of vimentin and fibronectin, as well as reduced levels of CDH1, while the levels of the regulators Twist and Snai1 remained unchanged.
Following the premise that both E6 and E7 of HPV have the ability to induce the expression of, bind to and stimulate the methyltransferase activity of Dnmt1
[76][77][185,189] and that E7 interacts with Mi2β, as well as with HDAC1 and HDAC2, to modulate the expression of cellular genes and viral genes by chromatin rearrangement
[78][79][157,213], and knowing that the cell lines HeLa (adenocarcinoma of the cervix), SiHa (squamous cell carcinoma) and Ca Ski (squamous cell carcinoma of the cervix) are representative of the most common types of CaCU with HR-HPV infection and have different viral load and different epithelial origin, a working group previously reported that HR-HPVs can induce a mesenchymal phenotype by negatively regulating the expression of CDH1 through different pathways in which E7, Snai1 and epigenetic mechanisms are involved
[80][214].