**3. HPV-Associated Lesions and Current Therapies**

HPVs are small, non-enveloped viruses, with a circular dsDNA genome of about 8 Kbp. The icosahedral capsid is composed of the major L1 protein and the minor L2 protein. Following infection of the epithelium basal cells, the E1 and E2 early proteins play key roles in viral DNA replication and amplification, as well as in regulating viral transcription. When actively expressed, the viral genome is maintained as an episome. Viral genome amplification, late gene expression, and viral progeny assembly are limited to terminally differentiated layers of the epithelium. Since terminally differentiated keratinocytes undergo growth arrest, HPV genomes have evolved, as a replication strategy, the expression of E6, E7, and E5 proteins, which are able to keep the infected cells in a competent state for DNA synthesis. Most HPV infections usually clear within 1–2 years but, in some cases, the virus may persist and occasional integration of its genome in the host's genome may occur. Consequently, the infected cells overexpress E6 and E7 due to the loss of the E2 transcriptional repressor coding sequence.

HPV infections are among the most common sexually-transmitted diseases and cause annually around 5% of all cancers worldwide [17–19]. However, only some genotypes are responsible for morbidity and mortality related to cancer, mostly of the cervix (CC) but also of the anogenital area and of the oropharynx, whose number is constantly increasing. According to the different oncogenic potential, HPVs are defined as high-risk (HR) types, which may cause the development of high-grade squamous intraepithelial (H-SIL) and cancer lesions, and low risk (LR) types, mainly causing anogenital warts. Worldwide, the HR HPV16 and HPV18 cause 71% of CC, with the remaining genotypes causing the residual HPV-associated cases [20,21].

Since more than 10 years, a bivalent vaccine against HPV16 and HPV18, and a quadrivalent vaccine that also targets the LR HPV6 and HPV11, responsible for genital warts, have been available. Recently, a nonvalent vaccine that offers protection against the five other most common HR genotypes (HPV 31, 33, 45, 52, and 58) and prevents about 90% of CC cases was developed [22]. The vaccines consist of DNA-free virus-like particles (VLPs) obtained by the only expression of the viral L1 protein through recombinant technology, and are administered with proper adjuvants. Nevertheless, effective prevention of the

HPV-associated pathologies is expected only in the long-term if HPV vaccination is able to reach a significant percentage of the target population worldwide [22–24]. The three HPV vaccines (bivalent, tetravalent, and nonavalent) have been licensed as prophylactic vaccines, but currently only 8% of low- and middle-income nations have introduced HPV vaccination programs in their health policies. In addition, in recent years many trials were performed to investigate the efficacy of these vaccines in preventing HPV disease recurrence after the treatment of high-grade cervical or anal lesions and condylomatosis, showing some promising results [24].

Of note, the 73rd World Health Assembly, held on August 2020, strongly encouraged the acceleration of actions aimed at eliminating cervical cancer as a public health problem. This should be pursued through "the inclusion of HPV vaccine into national immunization programs" and the "improvement of availability, affordability, accessibility, utilization and quality of screening, vaccines, diagnostics, and treatment and care of pre- and invasive cervical cancer" [23].

Treatment of HPV-associated lesions varies widely, mainly depending on lesion grade (high-grade lesions versus invasive cancer), lesion localization (lower genital tract, uterine cervix or head and neck), and tumour stage. In general, current therapeutic approaches aim at eliminating abnormal/malignant cells. This can mainly be achieved through surgery, chemotherapy, radiotherapy, targeted therapy, or immunotherapy. These approaches can be used alone or, in some cases, in combination.

In HPV-associated malignancies, E6 and E7 oncoproteins represent tumour-associated antigens, which are ideal targets for the development of vaccines stimulating specific cytotoxic T lymphocytes (CTLs). In HPV natural infection, E6- and E7-specific CD4 and CD8 immune responses are associated with the regression of HPV-cervical lesions. However, therapeutic vaccines are not yet available for clinical practice in spite of the numerous clinical experimentations carried out during the last 25 years, which used E6 and E7 through different platforms to raise specific immunity. The poor efficacy of these vaccines, due the lack of specific adjuvants of T-cell responses, has led to the interruption of many clinical trials in the early stages. Only recently, an improvement in efficacy was achieved by combining E6-E7 therapeutic vaccines with the use of radio- and chemotherapy (Cisplatin, Carboplatin, Paclitaxel), immune check-point inhibitors (anti-PD-1, anti-PDL1, anti-CTL4), cytokines (IFNalpha and IL-12), angiogenesis inhibitors (anti-VEGF), and modulators of tumour microenvironment (TME, Histone Deacetylase Inhibitors). Several new clinical trials are currently underway and the time for any authorizations has been further lengthened [24,25].

This context accounts for the huge bulk of research, particularly in the field of immunotherapy, still underway on therapeutic approaches against HPV lesions [26]. One of the promising treatments is undoubtedly that involving the use of intrabodies to block viral oncoproteins activities, and will be described below.

#### **4. Molecular Mechanisms of HPV-Induced Carcinogenesis**

HPV-associated carcinogenesis is known to be driven by the expression, in the transcription order, of the non-structural E6, E7, and E5 oncoproteins encoded by early genes [27].

The activity of E5, E6, and E7 has not been conclusively characterized and new functions are constantly being discovered. The following sections will briefly describe those molecular targets and biological activities that, causing the subversion of important regulatory pathways, are associated with the hallmarks of cancer as outlined by Hanahan and Weinberg [28]. In some instances, the role of the oncoproteins in determining a specific hallmark is well characterized, while in other cases, the precise activity connected with a hallmark has yet to be fully elucidated and the available lines of evidence provide only hints on the link between HPV oncoproteins and some hallmarks. A schematic representation of the HPV oncoprotein role in determining the hallmarks of cancer is shown in Figure 2.

**Figure 2.** Involvement of HPV oncoproteins in cancer hallmarks. Dysregulation of cell pathways ascribable to E6, E7, and E5 oncoproteins of HR HPVs is responsible for the entire spectrum of hallmarks of Human Papillomavirus (HPV)-related cancer (see text for details). In the figure, the involvement of each oncoprotein in the different hallmarks is reported.

#### *4.1. Cell Cycle Deregulation and Sustaining of Proliferative Signalling*

Deregulation of cell proliferation is one of the most characteristic traits of malignant cells. E7 oncoprotein targets key cellular mediators leading to uncontrolled cell proliferation. In particular, it establishes well-characterized interactions with the members of the "pocket proteins" family, represented by pRB, p107, and p130, and promotes their degradation [29]. This, in turns, allows the release of the E2F transcription factor, with the consequent expression of S-phase genes as well as of the p16INK4A cyclin-dependent kinase inhibitor. S-phase entry is also induced by E7 through the direct inactivation of essential regulators of G1-to-S-phase transition, such as p21 and p27 cyclin inhibitors. Sustained proliferation of HPV-infected cells is also induced by E5 oncoprotein, which increases the Epidermal Growth Factor Receptor (EGFR) signalling activity by forming an activating complex with it and impairing its degradation [30].

#### *4.2. Resistance to Cell Death*

Escape from cell death is one of the most important hallmarks of cancer, allowing cells with genomic defects to survive and continue to proliferate [31]. E6 interferes with several cell death pathways. It can abrogate apoptosis by promoting proteasomal degradation of p53 tumour suppressor, which controls the expression of pro-apoptotic genes [32]. E6 further promotes cell survival by impairing cell response to tumour necrosis factor (TNF), protecting the infected cells from Fas-induced apoptosis [33] and transactivating survivin gene promoter [34]. E5 oncoprotein also supports antiapoptotic activity during the first steps of the oncogenic process through downregulation of Fas-Ligand (Fas-L) on the cell membrane [33] and increased degradation of Bax [35].

#### *4.3. Avoiding Immune Destruction*

HPVs have developed mechanisms to counter the destruction of infected cells by the host's immune system. In this context, E5 plays an important role by promoting MHC class I retention in the Golgi apparatus, resulting in impaired viral antigen recognition [36]. Recently, E5 was found to be also involved in the increased expression of PD-L1 and inhibition of effector T cells, events that facilitate the immune evasion of HPV-infected cells [37]. E6 plays a direct role in modulating the immune response against HPV antigens by impairing Interferon (IFN)-mediated host defence in different and interrelated ways [38,39]. E7 also interferes with IFN signalling [40] and inhibits the Toll-like receptor-9 (TLR9) recognition in cooperation with E6 [41].

#### *4.4. Replicative Immortality*

To ensure the unlimited cell proliferation associated with carcinogenesis, E6 induces overexpression of human telomerase reverse transcriptase (hTERT), the catalytic unit of the human telomerase [42]. This occurs through both direct promoter activation and proteasomal degradation of its transcriptional repressor NFX1-91 through the E6/E6AP complex [43]. Constitutive expression of hTERT is also established through epigenetic mechanisms depending on alteration of the activity of histone methylases and demethylases [44], with E6 representing the main player.

#### *4.5. Induction of Angiogenesis*

HPV-infected cells derive sustenance and oxygen from the surrounding tissues thanks to the ability of the viral oncoproteins to induce angiogenesis. E6 and E7 regulate several angiogenesis modulators, including both inducers and inhibitors of this process. Both E6 and E7 are able to trigger the angiogenic switch by upregulating the expression of Vascular Endothelial Growth Factor (VEGF) respectively through direct [45] or E2F1-mediated [46] transcriptional activation of the angiogenetic factor gene [47]. In response to E6 and E7 expression, Interleukin-8 (IL-8), a major angiogenesis inducer, is also promoted [48]. On the other hand, expression of the thrombospondin-1 and maspin inhibitors of angiogenesis is indirectly perturbed by E6 as a result of E6-mediated degradation of p53, which positively regulates these inhibitors [48].

#### *4.6. Deregulation of Cellular Energetics*

Alterations of cell metabolism are among the earliest changes observed in cancer cells. Both the E6 and E7 oncoproteins contribute to the switch from oxidative to glycolytic cell metabolism, known as Warburg effect. Mechanisms by which HPV oncoproteins induce reprogramming of cell metabolism are reviewed in detail elsewhere [49]. E6 interferes with the expression of genes involved in the control of glycolytic metabolism through its interactions with p53 and c-Myc, while E7 promotes the glycolytic pathway by upregulating the expression of different glycolytic enzymes, such as hexokinase, involved in the first step of glucose metabolism, and by direct binding and activation of M2-pyruvate kinase. In addition, HPV16 E5 does not directly regulate glycolytic enzymes but contributes to the metabolic switch by activating the EGFR pathway that, in turn, promotes an enhancement of the glycolytic metabolic program [49].

#### *4.7. Invasiveness and Metastasis Induction*

The E6 encoded by HR HPVs plays a pivotal role in this cancer hallmark as it downregulates proteins modulating cell polarity and motility such as the scribbled planar cell polarity protein (SCRIB) involved in cell polarization and differentiation, and the membrane-associated guanylate kinases 1, 2, and 3 (MAGI-1, 2 and 3) [50]. Furthermore, E6-mediated functional modulation or degradation of adhesion effectors allows matrixindependent cell growth [51], resulting in enhanced motility and invasion of HPV-positive cells. E6/E7 contribute to the metastatic and invasive behavior of HPV-positive tumours by increasing the expression of different matrix metalloproteinases (MMPs) [52]. Both E6 and

E7 are involved in the epithelial–mesenchymal transition (EMT), crucial process for invasion and metastasis, by regulation of E-cadherin [53,54] and N-cadherin [55]. HPV-induced invasive cancer behavior has recently been correlated with E5 activity as well [56]. E5 is in fact able to upregulate the growth factor receptor MET, critical for tumour cell invasion, motility, and cancer metastasis, at both protein and mRNA level. Through this activity, E5 contributes to increase motility of the HPV-positive keratinocytes, and may thus promote metastasis of HPV-associated malignancies.

#### *4.8. Genome Instability*

The uncontrolled cell proliferation promoted by HPV oncoproteins facilitates the accumulation of genetic aberrations and genomic instability. E7 plays a central role in this process by inducing centrosome aberrations [57]. By increasing the activity of the cyclin-dependent kinase 2 (CDK2), E7 further leads to an augmented risk of genomic instability [58]. Concomitantly, a higher mutation rate is caused by E6 [59]. Altogether, E6 and E7 interfere with almost all the main actors of the cellular DNA repair pathway, thus reducing and delaying the removal of damages from the host cell genetic material. Another important hallmark associated with the expression of E6 and E7 is the interference with effectors of epigenetic modifications (e.g., DNA methyltransferases and histone modification enzymes). By influencing their activity, HPV oncoproteins may cause either the activation of oncogenes or the silencing of tumour suppressor genes [60].
