HPV viruses are divided into five genera according to the sequence of their genotype, known as α, β, γ, μ, and ν
. The HPV alpha and gamma groups infect skin and mucosal tissue, whereas the beta-, nu- and mu-subtypes infect cutaneous sites, even without clinical manifestations
. All of the 12 HPV genotypes that are classified as group 1 carcinogens belong to the alpha genus: HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59
. Indeed, alpha-HPVs are transmitted through sexual contact, and can be considered the leading group of causative agents of sexually transmitted infections globally
. Moreover, their incidence rises sharply after the first sexual intercourse
. As the natural immune response to this virus is weak and variable, one person may acquire different types of HPV infections
. Low-risk alpha HPV types causing benign genital warts or condylomata acuminata, as well as common and plantar warts, are also found
The main orchestrators of cellular transformation by HPV are the oncoproteins E6 and E7. They inactivate p53 and retinoblastoma protein (pRB), respectively, leading to the cell’s inability to control the cell cycle checkpoints correctly, and thus exacerbating cell proliferation
[38]. Significantly, the E7-dependent inhibition of pRB leads to the cell cycle S phase transition, promoting cell proliferation and viral transcription
[42][43]. Another critical aspect of E7 is that it binds to p21 and p27, which are proteins belonging to the cyclin-dependent kinase (CDK) interacting protein/kinase inhibitory protein (CIP/KIP), and are involved in regulating the cell cycle, which increases cyclin-dependent kinase 2 (CDK2) activity, collaborating with the cell entering the G1 to S phase
[44][45]. In a normal physiological condition, p53 would counteract the effects of exacerbated cell proliferation while activating the cellular DNA damage response (DDR) during viral DNA integration into the host genome, leading to the inhibition of cell growth and apoptosis
[46][47]. However, HPV E6 inactivates p53 by targeting its proteasomal degradation and forming a complex with the E3 ubiquitin-protein ligase E6-associated protein, E6AP
[48][49]. Moreover, it is also important to highlight the fact that high-risk E6 has been reported to bind the hTERT protein as well as the repeating DNA sequence of telomeric DNA, in addition to controlling telomerase activity
[50]. High-risk E6′s role in hTERT, telomerase, and telomeric DNA is thus multilayered, emphasizing its crucial and overlapping role in immortalization
[51].
Cells infected with HPV are able to stop infection by activating signaling pathways that result in the induction of anti-viral status and IFN type I secretion. However, both E6 and E7 display mechanisms of suppressing this response. Among other effects, E6 binds to IRF-3 (Interferon Response Factor-3), inhibiting its activity. E7 binds to IRF-1 and recruits HDAC (histone deacetylase) to the promoters that would be activated by IRF-1 but are suppressed due to E7 activity
[52]. Moreover, E6 and E7 can inhibit the STAT-1 and protein kinase R (PKR) pathways in infected cells
[53].
3. Proteases and HPV Carcinogenesis
Proteases, also known as peptide hydrolases, are found in all organisms (from viruses to vertebrates), and are classified as enzymes that can cleave peptide bonds
[54][55][56][57]. There are more than 400 proteases described in humans, and more than 14% have the potential to serve as drug targets for a variety of diseases
[58][59].
A critical aspect of the proteases is substrate specificity. Some proteases, such as trypsin, display broad specificity and are capable of cleaving many different substrates
[60][61][62][63]. Other proteases, such as the urokinase-type plasminogen activator (uPA), are selective and cleave a limited number of substrates
[57][63].
Proteases can be subdivided into two major groups: exopeptidases and endopeptidases
[64]. Exopeptidases are known to hydrolyze the substrate chain’s amino or carboxy terminal, and the conferred specificity is determined by the fragment size
[65][66]. Endopeptidases are proteases that can cleave the amino acids that are non-terminal, and the classification relies on the chemical group present in the catalytic domain. Six classes of endopeptidases have been described: (i) cysteine proteases, (ii) aspartic acid proteases, (iii) threonine proteases, (iv) glutamic acid proteases, (v) serine proteases, and (vi) metalloproteases
[67].
Proteases are known to participate in different physiological processes, from the degradation of proteins for recycling, to apoptosis, the cell cycle, skin desquamation, semen liquefaction, epithelial differentiation, the regulation of blood pressure, and homeostasis
[68][69][70][71]. It is essential to highlight the facts that proteases are synthesized as inactive zymogens, and that they have to be cleaved to be activated, which can occur irreversibly through post-translational modifications, co-factors ligation, and changes in pH, among others
[71]. Therefore, the activation of proteases is a very regulated process that prevents the uncontrolled activation of the enzymes in the cell
[67][72]. The dysregulation of some proteases’ expression can lead to the development of diseases, such as cancer. These enzymes are also involved in the degradation of the extracellular matrix and the activation of growth factors and pro-inflammatory mediators, which participate in malignant transformation and tumor progression
[73].
Serine proteases are a family of proteases characterized by the amino acids responsible for the catalytic activity in proteolysis, which are serine, aspartate, and histidine
[74]. This family of proteases is involved in different biological processes, such as epithelial barrier formation, skin desquamation, fertilization, embryonal development, cell signaling, and tissue morphogenesis
[75].
As part of this family, there is a subgroup composed of membrane-anchored serine proteases, which is divided into (i) GPI—serine proteases that are anchored in the plasma membrane by a glycosylphosphatidylinositol anchor, (ii) Type I—serine proteases that have a single pass domain in the plasma membrane located close to the C-terminus end, and (iii) Type II—serine proteases that are anchored in the plasma membrane and have an anchor sign located close to the N-terminus end
[71][76].
Matriptase, a type II transmembrane serine protease, is expressed in different epithelial tissues, such as the skin, gastrointestinal tract, lungs, kidneys, prostate, and mammary glands
[77][78]. This protease is responsible for activating uPA (urokinase plasminogen activator), which is related to cell adhesion and migration regulation, and the activation of growth factors and metalloproteases zymogens
[79][80]. Different studies have shown that when matriptase is less expressed, there is also less activation of uPA in the cells of ovary and prostate cancer
[81][82]. Matriptase is also responsible for activating the PI3K-Akt-mTOR pathway after the proteolytic activation of the hepatocyte growth factor precursor (pro-HGF), which can promote cell proliferation and decrease apoptosis
[76]. PAR-2 (protease-activated receptor 2), a receptor expressed in different cell types, is related to cell adhesion, the maintenance of the skin barrier, and inflammatory responses
[83][84]. One study has shown that the absence of PAR-2 inhibited the appearance of premalignant lesions and spontaneous or induced carcinogenesis in mice that overexpress matriptase in the basal layer of the epithelia, which highlights the importance of PAR-2 activation by matriptase in oncogenesis
[84]. It has already been described that matriptase is dysregulated in different types of epithelial cancers and, more specifically, carcinomas
[78][84][85][86][87][88][89].
Furthermore, matriptase is inhibited by the hepatocyte growth factor activator inhibitor-1 and -2 (HAI-1 and HAI-2), which are serine proteases inhibitors
[90][91]. HAI-1 is a type I transmembrane serine protease inhibitor encoded by the SPINT1 gene. One study has shown that HAI-1 is a potent inhibitor of hepsin, matriptase, and prostasin in HPV-positive cells (SiHa and HeLa). In cervical tissue analysis, HAI-1 expression was correlated with higher rates of tumor growth, the stage of the disease, stromal invasion, vaginal invasion, and lymph node metastasis
[92]. Moreover, patients exhibiting higher levels of HAI-1 exhibited decreased disease-free and overall survival
[92]. Similarly, analyzing cervical cancer specimens and the biological functions of HPV-positive cell lines, findings have indicated that a lower expression of HAI-2 in cervical cancer may be correlated with poor prognosis as well
[93].
Other subgroups of serine proteases are the ones that are secreted to the extracellular space, such as the kallikreins. Kallikreins are serine proteases that can be divided into (i) plasma kallikrein, with one member, KLKB1; and (ii) tissue kallikreins, with fifteen members, which have either tryptic or chymotryptic specificity
[94]. KLK proteases are found in almost every tissue, with different physiological functions, such as skin desquamation and seminal clot liquefaction, related with various cancers, Parkinson’s and Alzheimer’s diseases
[94]. The relationship between KLKs’ activation and different types of viruses, such as influenza and HPV, has been described
[95][96]. After HPV infection, the virus has to remove the capsid to expose the viral genome. The HPV16 virus can bind to heparan sulfate proteoglycans located in the host cell surface or the ECM
[97][98][99]. After the binding, the late gene 1 (L1) undergoes a conformational change, which leads the protein to be cleaved by Kallikrein 8 (KLK8)
[96]. Furthermore, the knockdown of KLK8 in HeLa and HaCaT cell lines has shown an inhibitory effect of HPV16 infection, while the irreversible serine protease inhibitor AEBSF [4-(2-aminoethyl) benzenesulfonyl fluoride] also had the same effect
[100]. The conformational change caused after L1 cleavage by KLK8 facilitates access to late gene 2 (L2) protein, which is found in the capsid lumen and facilitates the uncoating of the virus
[100]. In another study, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in cervical tissues, the researchers found that 95 proteins were dysregulated in the samples. Among those, the expression of ECM2 and the serine proteases KLK6 and MASP1 were increased in a stage-dependent manner. In particular, KLK6 was considered a highly significant prognostic marker, as it demonstrated a decrease in the overall survival (OS) and disease-free survival rates, showing that this protease may be considered a potential biomarker for the diagnosis and prognosis of cervical cancer
[101].
Metalloproteases (MMPs) are critical enzymes which are responsible for the degradation of the extracellular matrix (ECM)
[102]. These proteases participate in various physiological processes, especially in connective tissue remodeling, such as the postpartum involution of the uterus, ovulation, and wound healing, and in pathological processes such as joint destruction in rheumatoid diseases
[103][104]. It is known that metalloproteases are secreted as zymogens, and that they are activated proteolytically. Metalloproteases are divided into their respective subfamilies: collagenases (MMP-1, MMP-8, MMP-13), gelatinases (MMP-2 and MMP-9), stromelysins (MMP-3, MMP-10, MMP-11, and MMP-17), matrilysins (MMP-7 and MMP-26), membrane types (MMP-14, MMP-15, MMP-16, MMP-24, and MMP-25), and other types (MMP-12, MMP-19, MMP-20, MMP-21, MMP-22, MMP-28, and MMP29)
[66][105].
Studies of cervical tissues with cervical intraepithelial neoplasia and invasive squamous cell carcinomas (tested for HPV expression) have shown that the expression of MMP-2 in preinvasive lesions and MMP-1 and MMP-2 in invasive cancer suggests a gradual increase in the potential of cancer invasion
[102]. Furthermore, analyses of the expression of MMP-1 in cell lines (transformed with HPV18) and in clinical samples of cervical squamous cell carcinomas (SCC) have shown that MMP-1 is more expressed in SCC samples when compared with normal tissues, and that this protein can serve as a marker of the invasiveness of SCC
[106]. The production of MMP-9 was also up-regulated in cervical intraepithelial neoplasia (CIN 2 and 3) and in invasive carcinomas, which suggests a possible marker for early tumor progression
[107].
The E6 HPV viral oncoprotein can interact with PDZ domains, which are 80–110 residue-containing domains that are part of the signaling proteins’ C-terminal
[108][109]. The serine protease HTRA-1, which contains a PDZ domain in its C-terminal region, is expressed in various tissues, and is also associated with different pathologies, such as some types of cancer
[110][111][112][113]. One study has shown that the overexpression of the serine protease HTRA-1 is responsible for the prevention of cell proliferation in cervical HPV-negative cell lines and increasing cell proliferation in cervical HPV-positive cells, inferring that HTRA-1/E6 interaction is the underlying mechanism for the bypassing of growth arrest in HPV-positive cervical cancer cell lines
[114].
Another important group of proteases that are related with HPV carcinogenesis are the Ubiquitin proteases. USP46 is recruited to deubiquitinate and stabilize Cdt2/DTL by the E6 of high-risk HPV but not low-risk HPV
[115]. Cdt2—a component of the CRL4Cdt2 E3 ubiquitin ligase—is stabilized, which restricts the amount of Set8, an epigenetic writer, and promotes cell proliferation
[115]. USP46 is required for HPV-transformed cells to proliferate, but not for non-HPV cells to proliferate
[115]. Human cervical malignancies have a high level of Cdt2, and knocking down USP46 in xenografts stops HPV-transformed tumor growth
[115]. Oncogenic E6 recruits a cellular deubiquitinase to stabilize critical cellular proteins, and because the E6-USP46-Cdt2-Set8 pathway is important in HPV-induced malignancies, USP46 is a target for cancer therapy
[115]. Another study has shown that USP13 is essential for HPV-positive cervical cancer cells to proliferate, at least in part by deubiquitinating and stabilizing the prosurvival protein Mcl-1
[116]. Importantly, the pharmacological inhibition of USP13 sensitizes HPV-positive cervical cancer cells to BH3 mimetic inhibitors, implying that targeting USP13 could be beneficial in the treatment of these tumors
[116]. Furthermore, another study discovered ubiquitin-specific protease 15 (USP15) as an HPV16 E6-interacting protein using the yeast two-hybrid technique
[117]. HPV16 E6 polyubiquitin chains and/or ubiquitin precursors are cleaved by USP15, and could boost HPV16 E6 levels by preventing E6 degradation
[117]. The degradation of HPV16 E6 was reduced by USP15 in a dose-dependent manner; these findings imply that USP15, as a deubiquitinating enzyme, can stabilize E6 and, as an oncoprotein, can influence biological activities in infected human cells
[117]. Besides this, an important study highlighted that long-term hypoxia activates NF-κB, which is mediated via an effect of the HPV-encoded E6 protein on polyubiquitination and the subsequent degradation of the CYLD K63 deubiquitinase in HPV-positive cancer cells
[118].