Chapter I: General introduction

Short history of papillomavirus-induced carcinogenesis

Human papillomaviruses (HPV) are small non-enveloped double-stranded circular DNA viruses. Their genome is approximately 8 kb and is incorporated in an icosahedral capsid (Figure 1) [START_REF] Longworth | Pathogenesis of human papillomaviruses in differentiating epithelia[END_REF]. HPVs are epitheliotropic viruses that infect keratinocytes at a wide range of body sites. To date, based on their DNA sequence variation, 170 HPV types are identified [START_REF] De Villiers | Cross-roads in the classification of papillomaviruses[END_REF]. The most frequent manifestation of HPV infection is the development of warts. However, some HPV types are involved in the development of cancers. The research in HPV field is centered on the study of the relationship between HPV infection and cellular transformation.

Research on papillomaviruses (PV) was initiated about more than a century ago and passed through several phases. Few years after the use of filters retaining bacteria to demonstrate the filterability of the causative agent of tobacco mosaic disease [START_REF]Uber die Mosaikkrankheit der Tabakpflanze[END_REF], in late 19 th century, experimental transmission of papilloma in dogs was reported [START_REF] Mcfadyan | Note on the experimental "transmission of warts in the dog[END_REF]. These were the first observations suggesting viruses as causative agents. In 1907, Ciuffo reported the cell-free transmission of human warts, demonstrating for the first time a viral etiology for human disease [START_REF] Ciuffo | Innesto positivo con filtrado di verrucae volgare[END_REF]. A second phase of research in this field was stimulated by several observations related to the biology of PV in animals, in the 1930s. This period was initiated by the study of cottontail rabbits' papilloma in 1933. Extracts isolated from large warts of wild cottontail rabbits' lesions contained infectious particles capable of inducing cutaneous papilloma in domestic rabbits [START_REF] Shope | Infectious Papillomatosis of Rabbits : With a Note on the Histopathology[END_REF].

This virus is now known as the Cottontail rabbits PV (CRPV) and became an important experimental model of viral tumorigenesis. With the advent of electron microscopy, tobacco mosaic virus was observed first in 1939 and a decade later HPV particles visualization was achieved [START_REF] Strauss | Crystalline virus-like particles from skin papillomas characterized by intranuclear inclusion bodies[END_REF]. Although the research on papillomaviruses was hampered by the inability of these viruses to propagate in vitro, the interest on this field increased when Ito and Evans showed that papillomavirus DNA inoculation into rabbits was sufficient to induce carcinomas (1961) [START_REF] Ito | Induction of Tumors in Domestic Rabbits with Nucleic Acid Preparations from Partially Purified Shope Papilloma Virus and from Extracts of the Papillomas of Domestic and Cottontail Rabbits[END_REF]. This was the first demonstration that PV genome displays oncogenenic properties. Later on, Crawford and Crawford elucidated the physical properties of HPV DNA, in 1963 (9).

Following the revolution of molecular cloning in the 1970s, cutaneous HPV types were identified in malignant skin lesions in patients with a rare hereditary skin disorder, Epidermodysplasia Verruciformis (EV) [START_REF] Orth | Characterization of two types of human papillomaviruses in lesions of epidermodysplasia verruciformis[END_REF], providing the first link between HPV and human cancer. With the use of the new molecular biology technics, the plurality of HPV types was demonstrated and two mucosal low-risk HPV types (i.e. 6 and 11), and shortly after, two mucosal high-risk HPV types (i.e. 16 and 18) were isolated from genital warts in early 1980s [START_REF] Orth | Characterization of a new type of human papillomavirus that causes skin warts[END_REF][START_REF] Gissmann | Human papilloma viruses (HPV): characterization of four different isolates[END_REF][START_REF] Gissmann | Human papilloma virus DNA: physical mapping and genetic heterogeneity[END_REF][START_REF] Gissmann | Partial characterization of viral DNA from human genital warts (Condylomata acuminata). International journal of cancer[END_REF][START_REF] Gissmann | Molecular cloning and characterization of human papilloma virus DNA derived from a laryngeal papilloma[END_REF][START_REF] Boshart | A new type of papillomavirus DNA, its presence in genital cancer biopsies and in cell lines derived from cervical cancer[END_REF][START_REF] Durst | A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions[END_REF]. The initial studies aiming to demonstrate the role of HPV in human carcinogenesis was based on the hypothesis that only one type existed. This assumption led to many contrasting results when different studies analyzed lesions from different anatomical regions. The discovery of the existence of different HPV types with mucosal or cutaneous tropism explained the inconsistencies reported in many studies. Most importantly, these new findings overcame the criticism of the implication of HPV in cervical cancer proposed in 1976 [START_REF] Zur Hausen | Condylomata acuminata and human genital cancer[END_REF], and epidemiological study clearly identified HPV infection as main risk factor for cervical cancer [START_REF] De Villiers | Human papillomavirus infections in women with and without abnormal cervical cytology[END_REF]. In 1987, studies of Durst and colleagues showed in vitro experimental models the oncogenic properties of mucosal high-risk HPV16, and in particular highlighted the transforming abilities of the products of two early genes, E6 and E7 [START_REF] Durst | Molecular and cytogenetic analysis of immortalized human primary keratinocytes obtained after transfection with human papillomavirus type 16 DNA[END_REF]. Subsequently in last three decades, a vast number of studies have fully characterized the biological properties of E6 and E7 as main viral oncoproteins, being able to deregulate fundamental cellular events, such as cell cycle and apoptosis [START_REF] Zur Hausen | Papillomaviruses causing cancer: evasion from host-cell control in early events in carcinogenesis[END_REF]. Thanks to the knowledge built on HPV biology, prevention methods are developed to defeat cervical cancer [START_REF] Markowitz | Quadrivalent Human Papillomavirus Vaccine: Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR. Recommendations and reports : Morbidity and mortality weekly report[END_REF][START_REF] Priebe | 2012 cervical cancer screening guidelines and the future role of HPV testing[END_REF][START_REF] Vetter | Moving forward: human papillomavirus vaccination and the prevention of cervical cancer[END_REF]. Several screening strategies for HPV-induced cervical lesions have been put in place in many worldwide countries, determining a strong decrease of cervical cancer incidence. In addition, two prophylactic HPV vaccines are now commercially available, one quadrivalent that included two mucosal low-risk HPV (e.i. 6 and 11) and two mucosal high-risk HPV (e.i. 16 and 18) and one bivalent that includes two mucosal high-risk HPV (e.i. 16 and 18).

Although research was initially focused on the role of mucosal high-risk HPV types in cervical cancer, it is now well established that this group of viruses are the etiological factor of a subset of other genital tract cancers (e.g. vagina, vulva, anus, and penis) as well as head and neck cancers [START_REF] Zur Hausen | Immortalization of human cells and their malignant conversion by high risk human papillomavirus genotypes[END_REF][START_REF] Khan | Human papillomavirus detected in female breast carcinomas in Japan[END_REF][START_REF] Finzer | The role of human papillomavirus oncoproteins E6 and E7 in apoptosis[END_REF][START_REF] Ferris | Human papillomavirus-16 associated squamous cell carcinoma of the head and neck (SCCHN): a natural disease model provides insights into viral carcinogenesis[END_REF][START_REF] Fakhry | Clinical implications of human papillomavirus in head and neck cancers[END_REF]. HPV research is still expanding the aim to identify new links with additional human cancers; such has cutaneous HPV types and nonmelanoma skin cancer (NMSC) [START_REF] Bouvard | The cutaneous human papillomavirus types and non-melanoma-skin cancer[END_REF][START_REF] Pfister | High prevalence of epidermodysplasia verruciformis-associated human papillomavirus DNA in actinic keratoses of the immunocompetent population[END_REF].

Classification and phylogeny

The HPVs were originally included in the Papoviridae family, together with polyomaviruses.

Subsequently, due to morphological characteristics and the accumulated knowledge on PVs genome, a distinct family of Papillomaviridae was created. PVs are classified according to the species of origin and the degree of relationship between viral genomes. A recent phylogenetic tree has been designed groups the different PV types and at least 170 HPV types are presently identified (Figure 2) [START_REF] De Villiers | Classification of papillomaviruses[END_REF][START_REF] De Villiers | Cross-roads in the classification of papillomaviruses[END_REF]. The open reading frame of L1 gene is particularly conserved among PVs and is used for their classification. Phylogeny of PVs is organized with following taxonomic levels: family, genus, species, types, sub-types, and variants [START_REF] De Villiers | Classification of papillomaviruses[END_REF]. Papillomaviridae family is composed of 16 genera with less than 60 % homology (from alpha to pi). Each genus is divided into several numbered species (60 to 70 % homology) that are divided into types ( 71to 89 % homology). Subtypes differ with only 2 to 10 % and variant with less than 2 % differences [START_REF] Bernard | Genome variation of human papillomavirus types: phylogenetic and medical implications. International journal of cancer[END_REF]. Variants are indicated in reference to a prototype sequence of a PV gene and one nucleotide variation can be sufficient to define a variant.

HPV are represented by the 5 genera: alpha, beta, gamma, mu and nu. Based on their tissue tropism, two main groups of HPV have been identified: cutaneous and mucosal. According to their oncogenic potential, mucosal HPV types are divided into low-and high-risk. Some of the cutaneous types are preferably found in EV associated with a high risk of developing skin cancer upon UV-exposure.

Morphology and genomic organization

The HPV viral particle is approximately 55 nm in diameter and formed by an icosahedral capsid that is composed of L1 and L2 proteins arranged in 72 capsomers (Figure 1) [START_REF] Hagensee | Self-assembly of human papillomavirus type 1 capsids by expression of the L1 protein alone or by coexpression of the L1 and L2 capsid proteins[END_REF]. The number of L1 and L2 proteins was investigated and recent studies report that the capsid of HPV is composed of 360 L1 and 72 L1 molecules [START_REF] Buck | Arrangement of L2 within the papillomavirus capsid[END_REF][START_REF] Pereira | Insights into the role and function of L2, the minor capsid protein of papillomaviruses[END_REF]. L1 plays an important role in the formation of the capsid and L2 is mainly involved in the encapsidation of the viral genome [START_REF] Casini | In vitro papillomavirus capsid assembly analyzed by light scattering[END_REF].

The HPV genome is organized as illustrated in figure 3. The genome size is approximately 8 kb forming a chromatin-like complex by associating histones. It is divided in three regions: (i) the long control of about 1 kb, (ii) the early region of about 4 kb, and (iii) the late region of about 3 kb. The long control region (LCR) encodes the cis regulatory elements regulating replication and gene expression [START_REF] Longworth | Pathogenesis of human papillomaviruses in differentiating epithelia[END_REF][START_REF] Zur Hausen | Papillomavirus infections--a major cause of human cancers[END_REF]. The early region contains genes expressed during the initial phase of the virus life cycle and their products are important in genome replication and transcription as well as in processes of host cell transformation. The late region encodes the capsid proteins that are involved in the last steps of the virus life cycle. Transcription of each of the coding regions, early and late, is regulated through its respective promoter containing responsive elements for host-cell transcription factors. In undifferentiated cells only the early promoter is active whereas both early and late promoters are active in differentiated cells [START_REF] Wooldridge | Regulation of human papillomavirus type 31 gene expression during the differentiation-dependent life cycle through histone modifications and transcription factor binding[END_REF][START_REF] Hummel | Differentiation-induced and constitutive transcription of human papillomavirus type 31b in cell lines containing viral episomes[END_REF].

Despite the highly conserved structure of the genome, HPV types of different genera have some exclusive features. For instance, the beta HPV genome is relatively short in comparison to the mucosal HPV types, ranging from 7.4 to 7.7kb. This fact is due to the considerably reduced size of the LCR, around 400bp compared to 650-900bp in other papillomaviruses. Beta HPV types display some differences also in the coding region. Most of them lack the E5 gene, with the only exception of HPV14. In addition, the E2 ORF of all the beta HPV types is much longer compared to the other papillomaviruses. Also the mucosal high-risk HPV types present some specific characteristics. Indeed, HPV 16, 18 and 31 produce an additional early protein, E8^E2C that represses the expression of the viral oncogenes E6 and E7 and consequently cellular proliferation [START_REF] Fertey | Growth inhibition of HeLa cells is a conserved feature of high-risk human papillomavirus E8^E2C proteins and can also be achieved by an artificial repressor protein[END_REF].

Similarly to beta HPV genus, gamma HPV types lack the E5 gene. The isolation of three novel and related gamma HPV types (101, 103 and 108) from cervical tissues has been recently reported. Interestingly, the genomes of these three gamma HPV types lacks the E6 open reading frame [START_REF] Chen | Human papillomavirus (HPV) types 101 and 103 isolated from cervicovaginal cells lack an E6 open reading frame (ORF) and are related to gamma-papillomaviruses[END_REF][START_REF] Nobre | E7 oncoprotein of novel human papillomavirus type 108 lacking the E6 gene induces dysplasia in organotypic keratinocyte cultures[END_REF]. The numbers at the ends of each of the branches identify the Type; c-numbers refer to candidate types; all other abbreviations refer to animal papillomavirus types. The number at the inner semicircular symbols refer to the papillomavirus species and the outermost semicircular symbols identify papillomavirus genera. The tree was constructed using the MEGA5.1 program [START_REF] De Villiers | Cross-roads in the classification of papillomaviruses[END_REF].

HPV-associated malignancies

Cervical cancer

The alpha genus comprises approximately 30 HPV types that infect the mucosa of the genital and oral tract as well as several benign cutaneous HPV types that are associated with the development of common skin warts. Based on their oncogenic potential, the mucosal HPV types are divided in into two groups: low-risk HPVs (e.g. types 6 and 11) that are mainly associated with benign genital warts, and high-risk HPVs that are the etiological agents of cervical cancer [START_REF] Zur Hausen | Papillomaviruses and cancer: from basic studies to clinical application[END_REF]. In a recent monograph, the International Agency for Research on Cancer as classified as carcinogenic to humans 12 different HR HPV types, namely 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 (46). HPV16 and 18 are the most frequently found HPV types in cervical cancers worldwide, being detected in approximately 50% and 20% of the cases, respectively [START_REF] Munoz | Epidemiologic classification of human papillomavirus types associated with cervical cancer[END_REF][START_REF] Smith | Human papillomavirus type distribution in invasive cervical cancer and high-grade cervical lesions: a meta-analysis update. International journal of cancer[END_REF].

The mucosal HPV types are sexually transmitted and in the female genital tract the area adjacent to the border of the endocervix and ectocervix, known as transformation zone or squamocolumnar junction, appears to be the preferential site for infection. It is believed that micro-traumas occurring during sexual intercourse strongly facilitate the viral infection.

Approximately 80% of sexually-active women get a HPV infection during their life. In the majority of the cases, the HPV infections remain asymptomatic and are cleared by the immune system in a relatively short time (6-12 months). In a small number of cases, infection may persist, and after a period of latency, low and/or high-grade cervical intraepithelial neoplasia (CIN) develop, which may still regress or progress to an invasive cervical carcinoma [START_REF] Ostor | Natural history of cervical intraepithelial neoplasia: a critical review[END_REF].

Impairment of the immune surveillance strongly facilitates viral persistence and subsequently HPV-induced carcinogenesis.

Penile Cancer

Cancer of the penis is common in developing countries (10 % of cancers in men), and is at least ten times higher than in the developed countries and mostly affects men aged between 50 and 70 years [START_REF] Guimaraes | Penile cancer: epidemiology and treatment[END_REF]. Penile cancer has a multi-factorial etiology. The identified risk factors for the development of this cancer include poor hygiene, smoking, lack of circumcision, multiple sexual partners, and HPV infection [START_REF] Guimaraes | Penile cancer: epidemiology and treatment[END_REF][START_REF] Pow-Sang | Epidemiology and natural history of penile cancer[END_REF]. The investigation of HPV involvement in penile cancer development revealed a heterogeneous prevalence of high-risk HPV types. Thus not all penile cancer cases are HPV-related (prevalence of 46,9 %) [START_REF] Chaux | Developments in the pathology of penile squamous cell carcinomas[END_REF]. HPV detection in penile cancer revealed that HPV 16 is the most frequent HPV type associated to this cancer (60 %) [START_REF] Miralles-Guri | Human papillomavirus prevalence and type distribution in penile carcinoma[END_REF]. Histological analysis of the HPV-positive cases revealed that the basaloid and warty subtypes of penile carcinomas show the strongest association with the HPV [START_REF] Parkin | Chapter 2: The burden of HPV-related cancers[END_REF][START_REF] Palefsky | Human papillomavirus-related disease in men: not just a women's issue[END_REF][START_REF] Backes | Systematic review of human papillomavirus prevalence in invasive penile cancer[END_REF]. Penile cancer is generally treated through surgery; however this affects psychology and sexuality of the patient. Therefore, chemotherapy and radiotherapy are also implemented for penile carcinoma treatment [START_REF] Delacroix | Therapeutic strategies for advanced penile carcinoma[END_REF][START_REF] Rossari | Advances in penile cancer management[END_REF].

Anal cancer

Anal cancer represents 3 % of the colon and rectum malignancies and appears mainly at the age of 60 years [START_REF] Palefsky | Human papillomavirus and anal neoplasia[END_REF]. Rates of this type of cancer are continuously increasing (2 % per year) [START_REF] Johnson | Anal cancer incidence and survival: the surveillance, epidemiology, and end results experience, 1973-2000[END_REF].

This observation is at least partly attributed to the sexual behavior during the last decades. Anal cancer development is related to several risk factors including smoking, HIV-positivity, previous cancers (cervical, vulvar and vaginal cancers), sexual behavior, and HPV infection [START_REF] Ryan | Carcinoma of the anal canal[END_REF]. As is for the cervical region, the anal region is very susceptible to HPV infection and the lesions form anal intraepithelial neoplasia (as observed in cervical cancer) that can progress to squamous cell carcinoma (SCC). HPV DNA was detected in 46 to 94 % of all anal cancers [START_REF] Schiffman | Chapter 2: Natural history of anogenital human papillomavirus infection and neoplasia[END_REF].

In populations of HIV-positive men having sex with men, studies detected high-risk HPV types in approximately 90 % of the cases. According to the tumor extend, surgery and chemoradiotherapy are the most practiced therapies for anal cancer.

Head and neck cancer

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, it accounts for 3 to 4 % of all cancers [START_REF] Vuyst | Prevalence and type distribution of human papillomavirus in carcinoma and intraepithelial neoplasia of the vulva, vagina and anus: a meta-analysis. International journal of cancer[END_REF][START_REF] Jemal | Global cancer statistics[END_REF]. Tobacco and alcohol use are traditionally associated with HNSCC development. However, thirty years ago, HPV was also suspected to play a role in the development of HNSCC [START_REF] Syrjanen | Immunohistochemical demonstration of human papilloma virus (HPV) antigens in oral squamous cell lesions[END_REF]. It is now well demonstrated that high-risk HPV types are also involved in a sub-set of head and neck cancers (HNC).

Approximately, 25% of worldwide oropharyngeal carcinomas are linked to high-risk HPV infection, while the role of these viruses in HNC, such as oral cavity, larynx, and hypopharynx, appears to be considerably less significant [START_REF] Marur | HPV-associated head and neck cancer: a virus-related cancer epidemic[END_REF][START_REF] Halec | Biological evidence for a causal role of HPV16 in a small fraction of laryngeal squamous cell carcinoma[END_REF]. Among the high-risk HPV types, HPV16 is responsible for the majority (between 86-95%) of the HPV-positive oropharyngeal carcinomas [START_REF] Kreimer | Human papillomavirus types in head and neck squamous cell carcinomas worldwide: a systematic review. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF]. The higher prevalence of HPV16 in HNC in comparison to cervical cancer may be explained by intrinsic features of two distinct anatomical sites. In the oropharynx the immune system is much more active than the genital tract. Biological and epidemiological studies have shown that HPV16 is clearly more efficient in evading the host immune response than the other high-risk HPV types [START_REF] Hasan | TLR9 expression and function is abolished by the cervical cancer-associated human papillomavirus type 16[END_REF][START_REF] Schiffman | The carcinogenicity of human papillomavirus types reflects viral evolution[END_REF]. Thus, it is likely that HPV16 is more efficient than the other high-risk HPV types in establishing a persistent infection in the oropharynx, which is an essential step for the development of a malignant lesion.

Non-melanoma skin cancer

Non-melanoma skin cancer (NMSC) represents the most frequently diagnosed form of cancer among the Caucasian population, with continuing increase in incidence worldwide [START_REF] Stern | The mysteries of geographic variability in nonmelanoma skin cancer incidence[END_REF]. The majority of NMSC are basal cell carcinomas (BCC), accounting for about 80 % of NMSC cases, and SCC account for the majority of the remaining NMSC [START_REF] Preston | Nonmelanoma cancers of the skin[END_REF][START_REF] Silverberg | Cancer statistics[END_REF]. It is well established that exposure to sunlight or ultraviolet A and B (UVA and UVB) can cause NMSC upon DNA damage [START_REF] Tran | UV and pigmentation: molecular mechanisms and social controversies[END_REF]. Fair skin, blond hair and blue eyes and immune status are also recognized as risk factors for NMSC [START_REF] Oberyszyn | Non-melanoma skin cancer: importance of gender, immunosuppressive status and vitamin D[END_REF][START_REF] Motley | Multiprofessional guidelines for the management of the patient with primary cutaneous squamous cell carcinoma[END_REF][START_REF] Forslund | A broad spectrum of human papillomavirus types is present in the skin of Australian patients with non-melanoma skin cancers and solar keratosis[END_REF]. However, several studies indicate that HPV can be a co-factor favoring skin carcinogenesis, particularly beta HPV types. Genus beta comprises a large number of cutaneous HPV types that appears to be involved in the development of NMSC.

Forty-three beta HPV types have been isolated and fully sequenced so far [START_REF] Bernard | Classification of papillomaviruses (PVs) based on 189 PV types and proposal of taxonomic amendments[END_REF]. Initial evidence for the possible association of beta HPV types with skin cancer came from the isolation of the first beta HPV types, HPV5 and HPV8, in the skin of cancer-prone patients suffering from a rare autosomal recessive genetic disorder called Epidermodysplasia verruciformis (EV) [START_REF] Bouvard | The cutaneous human papillomavirus types and non-melanoma-skin cancer[END_REF][START_REF] Pfister | Chapter 8: Human papillomavirus and skin cancer[END_REF]. EV patients are highly susceptible to beta HPV type infections and develop disseminated pityriasis versicolor-like lesions and flat warts [START_REF] Jablonska | Epidermodysplasia verruciformis as a model in studies on the role of papovaviruses in oncogenesis[END_REF][START_REF] Majewski | Human papillomavirus-associated tumors of the skin and mucosa[END_REF][START_REF] Orth | Characteristics of the lesions and risk of malignant conversion associated with the type of human papillomavirus involved in epidermodysplasia verruciformis[END_REF]. These skin lesions arise early in life and in approximately 30-60 % of cases progress to multifocal SCC at sun-exposed regions. With the development of highly specific and sensitive detection methods, it became clear that beta HPV types are also abundantly present in the skin of normal individuals [START_REF] Astori | Human papillomaviruses are commonly found in normal skin of immunocompetent hosts[END_REF][START_REF] De Koning | Prevalence and associated factors of betapapillomavirus infections in individuals without cutaneous squamous cell carcinoma[END_REF]. Investigation of the genetic background of EV patients identified a susceptibility locus within the chromosome 17. Mutations in EVER1 and EVER2 genes were therefore reported. In EV patients, a total of seven and five loss of function mutations were isolated in EVER1 and EVER2 genes, respectively [START_REF] Ramoz | A susceptibility locus for epidermodysplasia verruciformis, an abnormal predisposition to infection with the oncogenic human papillomavirus type 5, maps to chromosome 17qter in a region containing a psoriasis locus[END_REF][START_REF] Ramoz | Evidence for a nonallelic heterogeneity of epidermodysplasia verruciformis with two susceptibility loci mapped to chromosome regions 2p21-p24 and 17q25[END_REF][START_REF] Ramoz | Mutations in two adjacent novel genes are associated with epidermodysplasia verruciformis[END_REF]. These genes encode transmembrane proteins and also termed TMC6 (for EVER1) and TMC8 (for EVER2). They were recently reported to play a role in the regulation of zinc homeostasis that can have an effect on the HPV life cycle [START_REF] Keresztes | TMC and EVER genes belong to a larger novel family, the TMC gene family encoding transmembrane proteins[END_REF][START_REF] Lazarczyk | Role of Zn2+ ions in host-virus interactions[END_REF]. However, at present, it is still unclear whether EVER1 and EVER2 genes are involved the immune response to HPV infection.

Epidemiological studies, using anti-HPV antibodies and/or viral DNA as markers of infection, have provided evidence that non-EV individual with history of skin SCC showed a higher positivity for beta HPV infections than controls [START_REF] Farzan | Cutaneous alpha, beta and gamma human papillomaviruses in relation to squamous cell carcinoma of the skin: a population-based study. International journal of cancer[END_REF][START_REF] Iannacone | Case-control study of cutaneous human papillomaviruses in squamous cell carcinoma of the skin. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF][START_REF] Karagas | Genus beta human papillomaviruses and incidence of basal cell and squamous cell carcinomas of skin: population based case-control study[END_REF][START_REF] Forslund | Cutaneous human papillomaviruses found in sun-exposed skin: Beta-papillomavirus species 2 predominates in squamous cell carcinoma[END_REF][START_REF] Bavinck | Multicenter study of the association between betapapillomavirus infection and cutaneous squamous cell carcinoma[END_REF]. The fact that impairment of the immune system in organ-transplant recipients (OTRs) strongly increases the risk of NMSC further supports the role of an infectious agent in skin carcinogenesis [START_REF] Boyle | Cancer, warts, and sunshine in renal transplant patients. A case-control study[END_REF][START_REF] Kiviat | Papillomaviruses in non-melanoma skin cancer: epidemiological aspects[END_REF][START_REF] Walder | The skin and immunosuppression[END_REF][START_REF] Walder | Skin cancer and immunosuppression[END_REF]. Accordingly, beta HPV infections were associated with an increased risk of NMSC in OTRs [START_REF] Proby | A case-control study of betapapillomavirus infection and cutaneous squamous cell carcinoma in organ transplant recipients[END_REF]. Several data indicate that beta HPV types are involved at early stage of skin carcinogenesis. For instance, quantitative type-specific PCR revealed that higher viral genome copy number can be detected in pre-malignant lesions, actinic keratosis, than SCC (100, 101). In addition, far less than one HPV DNA copy per cell is present in NMSC biopsies [START_REF] Pfister | Chapter 8: Human papillomavirus and skin cancer[END_REF]. It is therefore likely that the presence of the viral genome may not be required in the later stages of carcinogenesis. This may imply a need to consider a new scenario for the role of HPV in NMSC pathogenesis, such as a "hit-and-run" mechanism. In this case, HPV viral DNA is required at the initiation phase facilitating the accumulation of DNA damage induced by UV-irradiation, but not for the maintenance of the transforming state.

HPV replicative cycle

Virus entry

Studies on the mucosal HPV types have shown that the first step in HPV infection is the interaction of the viral capsid with the cytoplasmic membrane of cells at basal layer of the epithelium. This event is mainly mediated by the major capsid protein L1 that interacts with to heparan sulfonated proteoglycan (HSPG) on the cell surface (102, 103). It is also possible that the viral particles bind another component of the cellular membrane. In fact, it has been proposed that 6 integrin may act as secondary cellular receptor for HPV particles (104).

However, other studies showed that cells that do not express 6 integrin can be still infected by animal or human PV (102, 105). After binding of HPV16 particles to the cellular membrane, their internalization is mediated by a clathrin-dependent endocytic pathway (106). Additional findings indicate that other mucosal HPV types may use different endocytosis pathways (107).

It is highly likely that the minor L2 capsid protein also plays a role in membrane binding and cellular internalization. In fact, anti-L2 antibodies against specific linear epitopes are able to block the internalization of L1/L2 virus-like particles in in vitro assays (108-110). Surviladze and colleagues have recently presented evidence for a novel mechanism of viral entry. They observed that after binding to the cell surface, HPV16 particles are released as a soluble complex with HSPG and growth factors. The latter proteins mediate the interaction of the soluble complex with their cognate receptors facilitating the internalization of the viral particles (111). Endocytosis via the clathrin is characterized by the fusion of the endosome with another type of endosome (e.g. lysosome) and the endocytosis via the caveoline system can lead to a direct fusion between the endosome and the endoplasmic reticulum. The length of virus entry may involve several molecular events that are responsible for the transfer of the virion from first contact of the cellular surface to specific receptors that mediate the entry (112). For instance, the keratinocytes extra-cellular matrix component Laminin 5 was found to be required for the entry of HPV11 (113). Following virus release from the endosome, L2 protein mediates the transfer to the cellular nucleus (114,115). The N-terminus of L2 is cleaved within the endosomal compartment via the cellular protease, Furin, thus releasing an L2/genome complex into the cytosol (116). The L2/genome complex may then interact with Syntaxin 18, which ferries the complex to a perinuclear site (114,116). L2 can be translocated in the nucleus through its NLS (117). Following entry and uncoating, HPV genomes are replicated in the nucleus to about 100 episomal copies per cell.

Genome maintenance

Subsequent to its entry, the virus maintains a low-copy number of its genome in the host cell nucleus (118). The proteins E1 and E2 play important roles in the maintenance of the genome in episomal form and its segregation during cell division (118-120).

Genome amplification

The HPV genome is amplified in the suprabasal layer of the epithelium where keratinocytes undergo cell cycle arrest and differentiation. The E6 and E7 oncoproteins ensure a high proliferation rate of the cells by blocking cell cycle exit and inducing S-phase entry (128).

Although host cell differentiation is necessary for the next steps of the viral life cycle, E7 forces cell cycle re-entry in differentiating cells by targeting a pRb family member, p130 (129,130). Entry in differentiation process induces higher expression of the early proteins involved in replication (131). Therefore, this event induces a high production of viral genome that can be ranged at thousands per cells (128, 132).

Virion synthesis

The assembly and release of the virions are dependent on the expression of the late proteins L1 and L2 and loss of E6 and E7 expression in the upper layer of the epithelium (Figure 3) (134-136). L1 and L2 proteins form capsomers in the cytoplasm and translocate into the nucleus thanks to nuclear localization signals using keryopherins (137). Virus assembly occurs in promyelocytic leukemia protein (PML) nuclear bodies, also referred as nuclear domains 10 (ND10). All virus components are recruited to the ND10 via an important role played by the capsid protein L2 (138). The viral genome is recruited by E2 protein to the ND10 assembly sites (139). Following the packaging, virions undergo the last phase of their life cycle by releasing from the cells and escaping from the cornified envelop (140). During the virus life cycle, cells are forced to proliferate, accumulating DNA damage and deregulating their homeostasis. HPV persistence can lead to malignant lesions and even cancers for some cases.

Human papillomavirus E6 and E7 roles in carcinogenesis

One of the first pieces of evidence for the oncogenic role of E6 and E7 was provided by the analysis of cervical cancer-derived cell lines. In many of these cells, HPV genome was found integrated into the host genome. This integration generally leads to disruption of the majority of viral genes with only preservation of the E6 and E7 genes that are efficiently expressed (141).

Silencing the expression of E6 and E7 genes in cervical cancer cell lines results in rapid death (142). E6 and E7 are small proteins of approximately 150 and 100 amino acids, respectively.

They have the capacity to alter important functions of the host cells resulting in their immortalization. E6 and E7 have several strategies to evade immunesurveillance, to alter cell cycle control, and to inhibit apoptosis. In the normal virus life cycle, these functions allow viral persistence and replication but, as side effect, cause the accumulation of DNA damage and subsequent malignant transformation. Both viral oncoproteins exert these activities by directly interacting with a vast number of cellular proteins. Several E6 and E7 functions are shared with many HPV types, while some are exclusive features of E6 and E7 belonging to the high-risk HPV types. Among all HPV types, the high-risk HPV16 is the most investigated and therefore many of its E6 and E7 functions have been fully characterized. In the contrary, only few studies have investigated the E6 and E7 properties from beta HPV types (143).

Major transforming activities of E6 and E7 from alpha HPV types

High-risk HPV E6 properties

The best characterized activities of HPV16 E6 and E7 are their ability to target tumor suppressors, p53 and pRb respectively. The functions of E7 in the host cell cause an abnormal increase in DNA replication resulting in cellular stress. In this situation the host cell would activate apoptosis through p53; however, E6 protein from high-risk HPV types is able to inhibit the p53-dependent programmed cell death. To counteract the cellular response to the E7mediated unscheduled proliferation, E6 binds p53 and causes its proteasome-dependent degradation (143).

The interaction between E6 and p53 was the first activity described for high-risk HPV E6 proteins (144). In a normal situation, cells maintain very low levels of p53 through the ubiquitin ligase mdm2 that is able to directly interact with p53 and to promote degradation via the proteasome pathway (145-147). Upon cellular stresses, several events lead to the loss of mdm2/p53 interaction, with strong stabilization of p53, which in turn promotes cellular arrest or apoptosis (148). In HPV16-infected cells, The E6 oncoprotein mediates the interaction of p53 with another ubiquitin ligase enzyme, E6 associated protein (E6AP), promoting the degradation of the tumour suppressor (149, 150). Thus HPV-positive cells display a relatively low expression of p53-regulated genes upon genotoxic stress, facilitating the accumulation of DNA damage and, ultimately, cellular transformation (151, 152).

In addition, E6 has developed other mechanisms to inhibit p53 transcriptional activity. It has been shown that E6 binds the transcriptional coactivators p300/CBP (CREB binding protein) that play a positive role in p53-regulated transcription via p53 acetylation (153-155). E6 protein inhibits another p53 coactivator, hADA3, that is part of acetyltransferases complex (156). This protein is degraded through an E6/E6AP-dependent process (157). Decreased levels of hADA3 correlate with the inhibition of p14ARF/p53 pathway and subsequent cellular immortalization of mammalian epithelial cells (158). G protein pathway suppressor 2 (GPS2) is another E6targeted transcription coactivator, that is known to inhibit RAS-and MAPK-mediated signaling (159). GPS2 protein is degraded upon interaction with E6 (159). In addition to affecting p53induced apoptosis E6 has also an effect on the mitochondrial-and death receptors-related apoptosis. E6 was shown to bind, together with E6AP, the protein Bak leading to its degradation (160). Bax mRNA levels are also affected by E6, downregulating its protein levels (161).

Moreover, E6 inhibits tumor necrosis factor (TNF) signaling. It binds to the TNF-receptor 1 and blocks the interaction with Fas associated death domain (FADD) (162-164).

High-risk HPV E7 properties

E7 proteins from high-risk HPV types can interact with pRb, p107, and p130 proteins, also termed pocket proteins (165,166). The protein E7 is able to induce proteasome-dependent pRb degradation. During the G1/G0 phase of uninfected cells, the pocket proteins interact with the transactivating domain of E2F transcription factors (167). Upon exposure of the cells to proliferative stimuli, pRb is phosphorylated by the G1 cyclin-dependent kinase (CDK) complexes and loses the ability to interact with E2F. In turn, free E2F drives the transcription of key genes that encode proteins involved in cell cycle progression (167). In HPV-infected cells, E7, via degradation of pRb, induces constitutive activation of the G1/S transition, and induces the cellular proliferation independent of external proliferative stimuli (168-170).

HPV16 E7 was reported to bind the ubiquitin ligase cullin 2, and this interaction is important for cellular transformation and cell cycle checkpoints deregulation. These observations suggest the contribution of cullin 2 in E7-mediated pRb destabilization (171).

Transition from G1/S to the S phase is also achieved by the induction of cyclin A/CDK2 and as reported for E6, E7 also has redundant mechanisms for deregulation of the cell cycle control proteins (172-174). Indeed, E7 is able to directly associate with two CDK2 inhibitors, p21Cip1 and p27Kip1, with consequent block of their functions (175,176). Beyond the role as regulator of cell cycle, it has been shown that pRb is involved in other cellular events, e.g. differentiation.

Therefore, the E7-mediated pRb degradation also results in inhibition of other cellular functions.

E7 from HPV16 has also been reported to deregulate the transcription of co-activators p300, CBP, and p/CAF (p300/CBP-associated protein) (177-179). These proteins are involved in the activation of E2F-mediated gene transcription (180). Similarly, E7 binds to histone acetyl transferases (HAT) and increases the levels of histone H3 acetylation within E2F-target gene promoters correlating with active transcription (181). In addition to binding pRb, E7 acts also on the histone deacetylases (HDAC) which remove acetyl groups from histones resulting in the formation of heterochromatin. High-risk HPV E7 binds to HDAC through the intermediate binding of Mi2 , a chromatin remodeling protein (182). The C-terminal domain of E7 plays an essential role in this interaction. Sequestration of HDAC by E7 results in the induction of gene expression following chromatin remodeling due to histone acetylation. Blocking the interaction of HDAC and pRb with E7 results, respectively, in the destabilization of viral genome maintenance and cell cycle progression during differentiation (182)(183)(184)(185). This demonstrates the importance of pRb and HDAC binding and inactivation by E7 during the process of keratinocytes immortalization.

Deregulation of NF-B pathway

Nuclear Factor-B (NF-B) constitutes a family of transcription factors: NF-B1 (p50 and its precursor p105), NF-κB2 (p52 and its precursor p100), RelA (p65), RelB and cRel (Rel). These proteins can form heterodimer or homodimers. The NF-κB protein family shares a highly conserved N-terminal region, known as the Rel homology domain (RHD), which is involved in dimerization, DNA binding sequence, interaction with the inhibitory IκB proteins, and contains a nuclear localization sequence (NLS). In a latent state, NF-κB dimers are mainly localized in the cytoplasm, sequestrated by binding the inhibitory subunits IκB. As NF-κB, IκB proteins form a family containing several members. In mammals, it includes IκB , IkBβ, IκBγ, IκBε, Bcl-3, and the precursor Rel-proteins, p100, and p105. Activating stimuli mediate the 26S proteasome-dependent degradation of IκB, following its phosphorylation by IκB kinases (IKK) at two conserved serine residues (S32 and S36) in its N-terminal regulatory domain. Then, NF-κB dimer is free to translocate to the nucleus, it binds specific κB sites and regulates the expression of a target gene (186).

As shown in figure 4, the canonical or classical pathway is characterized by the NF-κB dimer p50 and p65. Its activation is regulated by the IKK complex which is composed of the regulatory subunit IKKγ (NEMO) and the catalytic subunits IKKα and β. Following stimulation by different agents such as TNF-α or IL-1, viruses, genotoxic agents, and ionizing radiation, this pathway leads to the expression of several genes crucial for the activation of innate NF-B canonical pathway is characterized by the p50/p65 NF-κB subunits which are inhibited by IκB. The phosphorylation of IκB by CK2, IKKε or IKK complex leads to its degradation. This pathway targets genes implicated in inflammation and apoptosis. The non-canonical NF-κB pathway involves the p100/RelB complex which is targeted by the IKKα homodimer. Phosphorylation of p100 causes its processing to the active form p52. The activation of this pathway is mainly responsible for the expression of genes involved in B cell development NF-B signaling is found activated in many cancers and targeted by oncogenic viruses including EBV, HPV, and HTLV (190)(191)(192)(193). These viruses have developed several strategies to take advantage of the ability of NF-B to induce for instance anti-apoptotic and growthpromoting genes. In correlation with the presence of HPV, the NF-B pathway is found overexpressed in penile and cervical cancer cases (194)(195)(196). In laryngeal SCC cells infected with HPV, a positive correlation has been reported between the level of HPV16 E7 and the nuclear localization of the p65 subunit of . A study carried out on oral tissue biopsies, showed that in high-risk HPV positive tumors the positive transcriptional regulatory complex p65/p50 is more abundant than the homodimer complex p50/p50, which is often detected in HPV-negative cancers (197,198). Interestingly, in airway epithelium, it was observed that HPV16 E6-expressing cells have an increased transcription of the NF-Bregulated anti-apoptotic gene cIAP-2 (193). E6 induces this event via the induction of the nuclear binding activity of p52 and it requires the E6 PDZ binding motif (193). This function of E6 is important to protect HPV infected cells from TNF-induced apoptosis. Furthermore, expression of E6 and E7 in cervical keratinocytes leads to pro-proliferation-specific activation of NF-B (199). In contrast with these observations, some studies have reported that HPV16 oncoproteins can reduce NF-B signaling. HPV16 E6 was shown to attenuate p65 transcriptional activity and E7 to target IKK complex and reduces NF-B activity (200,201).

They inhibit interferon signals and their target genes. These effects of HPV oncoproteins could be important to counteract the immune sensing of the infected cells, and these functions could be in line with the evasion of the immune system allowing the virus to persist in the host cells.

Deregulation of hTERT

Telomeres' length is shortened after each cell cycle causing, at the Hayflick limit, genomic instability and cellular senescence. A normal cell undergoes apoptosis before reaching this limit, but tumor cells bypass this limit and proliferate continuously (202). Different mechanisms of maintaining telomere length have been described. E6 was shown to activate the catalytic subunit human telomerase reverse transcriptase (hTERT) of the ribonucleoprotein complex responsible for the synthesis of telomere repetitions (203)(204)(205). Several studies have shown that high-risk HPV E6 can activate telomerase activity by independent mechanisms. For instance, E6 induces hTERT transcription via activation of c-Myc which binds on the promoter of hTERT and positively regulates its transcription (206,207). In addition, E6, via the interaction with E6AP, promotes the degradation of a negative regulator of hTERT transcriptiom, nuclear transcription factor X-box binding 1-9 (NFX1-91) (208,209). Another splice form of NFX1, NFX1-123, is not affected by E6 and E6AP. This form of NFX1 acts positively on hTERT levels by increasing its mRNA stability (210,211).

Deregulation of PDZ proteins

Another well characterized property of E6 from high-risk HPV types is the ability to target several members of the membrane-associated guanylate kinase (MAGUK) family that are involved in the regulation of cell-cell contact via the tight junction and cell polarity (212-214).

They contain various protein/protein interaction domains, including PDZ motifs. The term PDZ refers to the first letters of the three first proteins in which a consensus domain of approximately 90 amino acids was identified. These proteins are: PSD (PostSynaptic Density), DLG (Drosophila disc large protein), and ZO1 (Zonula Occludens 1) (166).

The high-risk HPV16 E6 oncoproteins have a four amino acid PDZ-binding motif at the Cterminus that mediates the interaction with the MAGUK family members. The PDZ-binding motif of E6 protein is important for its transforming activity. Mutation of this motif results in the inhibition of cellular growth and viral DNA replication (215-218). Upon interaction, E6 induces the degradation of PDZ proteins leading to the loss of control of pathways that regulate cellular adherence and polarity (214, 219). Multiple PDZ proteins have been reported as E6

substrates including hDlg1, and hScrib (214,220,221). These events could increase the metastatic dissemination of the HPV-infected cells.

Major transforming activities of E6 and E7 from beta HPV types

As mentioned above, the molecular mechanisms and role of beta HPV types in skin carcinogenesis are still not sufficiently investigated. However, several initial studies provide evidence that E6 and E7 from specific beta HPV types can deregulate important cellular functions and lead to cellular immortalization. In particular, it has been shown that E6 and E7 from two beta HPV types, 38 and 49, are able to efficiently immortalize primary human keratinocytes (222)(223)(224)(225). These observations support the putative etiological role of beta HPV types in NMSC development.

Animal models

The implication of cutaneous beta HPV in skin cancer was investigated in animal model through the expression of several early genes in the skin using tissue-specific promoters such as keratin 10 (K10) or keratin 14 (K14) promoters. Transgenic mice expressing HPV8 E2, E6, and E7 proteins in their skin display a higher risk to developing NMSC (226)(227)(228)(229). Another study has shown that the expression of HPV20 E6 and E7 proteins in mice skin induces cutaneous cells hyperproliferation and inhibition of cell differentiation upon UVB-irradiation.

Similar observations were made with the expression of HPV38 E6 and E7. The exposure of transgenic mice to UVB with a skin-specific expression of HPV38 E6 and E7 leads to hyperproliferation and p21 transcription inhibition (230,231). The exposure of the same transgenic mice to chemical carcinogens, dimethylbenzanthracen (DMBA) and trimethylamin (TPA), enhanced the development of cutaneous epidermoid carcinomas compared to the control mice (230,232). Another study has shown that transgenic mice expressing HPV8 E2 or E6

protein developed skin tumors spontaneously or upon 233).

All together, these findings demonstrate the carcinogenic capacity of some beta HPV early proteins in in vivo models. This risk of tumor development in the presence of the cited viral proteins appears to act in synergism with other carcinogens.

Deregulation of p53 pathway

In contrast to E6 from mucosal alpha HPV types, those from cutaneous beta HPV types did not show the ability to degrade p53. However, a recent study has pointed out the destabilization of p53 protein levels in human keratinocytes expressing HPV49 E6 and E7, in line with their ability to immortalize human primary keratinocytes ( 224). The protein E6 from this beta HPV type appeared to bind p53 together with E6AP. The formation of this complex leads to p53 proteolysis that is not as efficient as with HPV16 E6 suggesting possible differences in this process between the two HPV types ( 224). HPV49 E6 is, so far, the only beta HPV protein shown to act directly on p53 protein stability. In addition, E6 from HPV23 was shown to block p53 activation by preventing its phosphorylation. HPV23 E6 binds to HIPK2 and hampers its interaction with p53, necessary for its phosphorylation at Ser46 (236). This activity of HPV23 E6 in human keratinocytes is important for the survival of the cells upon UV-exposure (236). Cutaneous HPV E6 is also able to inhibit the UV-induced apoptosis in the presence of high levels of active p53 protein (237).

Apart of acting directly on p53 function, E6 can inhibit apoptosis independently of p53. It has been shown that E6 interacts with the pro-apoptotic protein Bak which is involved in mitochondria-mediated induction of apoptosis for instance upon UV-exposure (238). In cells irradiated with UV and expressing E6 from HPV5, and other cutaneous HPV types of different genera (HPV10 and 77), Bak-mediated apoptosis is inhibited (238). This protein was shown to be degraded through ubiquitin-ligase interaction with E6 from cutaneous HPV5, 8, 10 and 77 (238). These features of beta HPV E6 proteins to block UV-mediated apoptosis reinforce the hypothesis of a possible synergism between HPV and UV-induced DNA damage during skin cancer development. In line with the inhibition of UV-induced apoptosis, E6 was moreover shown to affect DNA damage repair pathways. This results in the accumulation of DNA damage and double strand breaks leading to chromosomal instability. UV-exposure of the cells induces the formation of cyclobutane pyrimidine dimers that are the predominant DNA lesions in human skin cells. HPV5 E6 is able to affect the repair of this DNA damage when expressed in epidermal cells (239). The XRCC1 protein, implicated in single strand DNA breaks repair, has been also reported to be targeted and inhibited by HPV8 E6 (240).

Deregulation of pRb pathway

The knowledge on cutaneous beta HPV E7 proteins is extremely limited compared to those from mucosal alpha HPV types. The most relevant finding for cutaneous beta HPV E7 to mention is the characterization of some biological properties of HPV38 E7. It has been reported

that this protein is able to interact with pRb protein, as efficiently as seen for HPV16 E7, in human keratinocytes (222,224). This function of HPV38 E7 deregulates cell cycle check points in G1 and S phases, contributing to the transformation of the host cell ( 222). The observe effect on pRb by HPV38 E7 does not occur in presence of beta HPV20 E7 proteins (224). A comparative study of the effects of E7 proteins from several beta HPV types highlighted the efficiency of HPV38 E7, and HPV49 E7, in deregulating the pRb pathway. The expression of HPV49 E7 in human keratinocytes appeared to inhibit the inactivation of E2F by pRb and enhanced the expression of cell cycle regulators such as cMyc, cyclin A, and Cdc2 (224).

Deregulation of NF-B pathway

As exposed above, despite the important place of NF-B pathway in cellular homeostasis through its involvement in several functions such as inflammation and cellular growth balance, very few studies have investigated its deregulation upon the expression of beta HPV proteins.

A recent study has investigated the effect of E7 proteins from several cutaneous HPV types in U2OS cells, including the E7 proteins from beta HPV20, 37, 38, 92, 93, and 96 (241). In these cells, the investigators have reported the inhibition of NF-B activity in presence of E7 proteins that are able to bind IKK but not IKK . This finding is in line with what has been shown for HPV16 E7 in the same cellular model ( 200). Additional experimental analysis will be required to understand the effect of NF-B deregulation. In the present thesis we will explore the status of NF-B pathway in human keratinocytes expressing HPV38 E6 and E7.

Deregulation of hTERT

It

Deregulation of Notch pathway

Notch was described to play an important role in keratinocyte differentiation and cell cycle arrest processes (243). In human keratinocytes, Notch has been reported to act as a tumor suppressor (244). Several studies investigated the status of Notch signaling in presence of beta HPV proteins. It has been shown that E6 from several beta HPV types are able to inhibit Notch signaling (245). E6 proteins from HPV8 and as well as cutaneous HPV1 associate mastermindlike 1 (MAML1), a NOTCH coactivator, and repress the expression of Notch target genes (246,247). These findings highlight the potential important role of Notch pathway inhibition during HPV infection and the need of further characterization of the molecular mechanism.

Aim of the thesis

Viral infections trigger numerous host cell defenses. A well-known cellular protection against viral infection and spread is the suicide of the infected cells. To complete the viral life cycle, several viruses adopt complex strategies to evade apoptotic cell death. The molecular mechanism of high-risk HPV E6 and E7-mediated targeting of important cellular pathways such as p53 signaling is well described. However, knowledge on beta HPV E6 and E7 functions toward cellular transformation is still poor. In this thesis we have studied HPV38 E6 and E7

activities in deregulating p53 and NF-B pathways.

First study:

It has been shown that p53 functions are inhibited in HPV38 E6 and E7-expressing keratinocytes through the accumulation of Np73 . In this study we have investigated the individual contribution of E6 and E7 in IKK -nuclear accumulation and Np73 stabilization.

We have also further characterized the cooperation between IKK and Np73 to achieve the transcriptional inhibition of p53-regulated genes.

Second study:

In contrast to high-risk HPV types that are known to deregulate NF-B, we have limited knowledge about the influence of beta HPV early proteins expression on this pathway. In this study we have investigated the effects of HPV38 E6 and E7 proteins on the activity of NF-B signaling. We have also evaluated the role of NF-B in E6 and E7-mediated apoptosis inhibition. Finally we asked which of E6 and E7 is responsible of NF-B activation and through what potential mechanism.

Chapter II: Results 1

The E7 oncoprotein from beta human papillomavirus type 38 induces the formation of an inhibitory complex for a subset of p53-regulated promoters

1/ HPV38 E6 and E7 oncoproteins expression directly influence NF-B transcriptional activity

To determine the impact of HPV38 E6 and E7 expression on the NF-B pathway, we have analyzed the status of key players of this pathway in primary keratinocytes transduced with an empty retrovirus or with recombinant retroviruses expressing E6 and E7 from HPV10, 14 or 38. E6 and E7 from HPV10 and HPV14 do not display any in vitro transforming activities, while HPV38 E6 and E7 are able to efficiently immortalize primary human foreskin keratinocytes (38E6E7 HFK). The levels of phosphorylated I B (serine 32 and 36) and p65

(serine 536) were determined by immunoblotting in primary cells or cells expressing E6 and E7 from three different HPV types. As shown in Figure 1A, higher levels of phosphorylated I B and p65 were detected in 38E6E7 HFK in comparison to primary keratinocytes or keratinocytes expressing HPV10 or HPV14 E6 and E7. To corroborate these findings, we investigated whether HPV38 oncoproteins have the ability to modulate IKK complex activity.

To this end, 38E6E7 HFK constitutively expressing a pRetrosuper (pRS) containing shRNA for HPV38 E6 and E7 were generated. Efficiency of E6 and E7 expression inhibition was measured by quantitative PCR (Figure 1D). IKK was then isolated by immunoprecipitation from HPV38 pRS38E6/E7 and from HPV38 pRS keratinocytes and incubated with GST-I B substrate in presence of [ -32P]ATP. The in vitro kinase assay showed a higher kinase activity of IKK in presence of the viral proteins supporting the previous result (Figure 1B).

Consistently, in the same cellular system, we observed a reduction in the protein levels of phospho-I B and phospho-p65 in presence of the shRNA for HPV38 E6 and E7 (Figure 1C).

Finally, we verified the transcriptional activity of NF-B in the same cells by measuring the mRNA levels of xIAP, cIAP1 and cIAP2 transcripts, products of NF-B anti-apoptotic target genes. As expected, we observed lower expression levels of xIAP, cIAP1 and cIAP2 genes in the pRS38E6/E7 cells compared to the cells harboring the pRS empty vector (Figure 1D). These results show that NF-B pathway is up-regulated in 38E6E7 HFK through the induction of the kinase activity of IKK . A/ Immunoblot on total proteins from cellular lysates of HFKs expressing E6 and E7 oncoproteins from HPV10, 14 or 38 or transfected with the pLXSN empty vector. Antibodies were used to look for the expression of phosphorylated-IkBa and phosphorylated-p65 compared to the total I B and p65 protein amount. B/In vitro kinase assay using same amounts of immunoprecipitated proteins from 38E6/E7HFKs expressing pRS38E6/E7 or with the empty pRS vector (left panel). IKK proteins were incubated with GST or GST-I B during 30min at 30°C in presence of [g-32P]ATP and radio activity is then detected. C/ HPV38 E6/E7HFKs expressing pRS38E6/E7 or pRS ampty were used to detecte by immunobloting the protein levels of phopho-I B and phospho-p65 compared to the total amounts of this proteins. D/ Quantitative PCR on the total cDNA obtained with RT-PCR on total RNA isolated from 38E6/E7HFKs expressing pRS38E6/E7 or containing the empty pRS vector. The expression levels of HPV38E6, HPV38E7, xIAP, cIAP1 and cIAP2 were analysed and normalized on the GAPDH expression level.

2/ E7 oncoprotein plays an important role in HPV38-mediated up-regulation of NF-B pathway

Next, we asked whether the activation of IKK activity was due to the expression of E6 and/or 38E7 than in the one expressing 38E6, supporting a major role of HPV38 E7 in the events described so far (Figure 2).

3/ IKK physically interacts with HPV38 E7 and is translocated into the nucleus

It has been previously shown that certain viruses constitutively activate NF-B using different strategies. HTLV-1 Tax oncoprotein was shown to interact with the IKK complex by direct binding of NEMO (IKK ). Therefore, we asked whether similarly to Tax protein, HPV38 E7 could target the IKK complex. To explore this hypothesis, we first investigated IKK and IKK sub-cellular localization performing immunofluorescence staining on 38E6E7 HFK. Previous findings have shown that IKK is localized in the cytoplasm, while IKK is also cytoplasmic but with a shuttling capacity between the cytoplasm and the nucleus. In addition, studies on E7 from different HPV types indicate that the viral protein can shuttle between the nucleus and cytoplasm. We analyzed IKK and IKK localizations in 38E6E7 HFK, and the result shows that in contrast to IKK , which appeared cytoplasmic, IKK was localized in the nucleus (Figure 3A). In agreement with our previous results (Chapter I, Results I), we found that IKK nuclear localization was associated with HPV38 E7 expression. Indeed, HPV38 E6 expressing HFK showed a similar IKK cellular localization to primary HFK, (Chapter II, Results 1). Since HPV38 E7 and IKK appeared to co-localize we asked whether they physically interact. We therefore transiently transfected Human Embryonic Kidney (HEK) 293 cells with Flag-38E7 and un-tagged IKK , and cellular extracts were prepared for immunoprecipitation of Flag-38E7

or IKK . The immunoblotting analysis shows that the two proteins co-immunoprecipitated, indicating a physical interaction between HPV38 E7 and IKK (Figure 3B). This result correlates with the IKK translocation in the nucleus in presence of HPV38 E7 protein.

4/ IKK is localized in the nucleus of several cancer cell lines

The studies on HPV38 E6 and E7 HFK highlighted a key role of IKK nuclear localization in cellular proliferation. To determine the role of IKK in additional transformed cells, we binding the regulatory subunit of the IKK complex (IKK ) (248, 249). Therefore, it is possible that the interaction of E7 with IKK could lead to the stimulation of its kinase activity and consequent stimulation of NF-B pathway. The E7 protein domains involved in the interaction with IKK are still not identified. Their characterization is important for the understanding of the biological significance of this protein complex. Indeed, generation of HPV38 E7 mutants unable to bind IKK will provide evidence whether the interaction with the cellular kinase is important for the stimulation of its activity.

In our study we have focused on a small number of NF-B-regulated genes, but it is likely that a larger number of cellular genes are deregulated. Broader investigation of HPV38-deregulated NF-B target genes expression will help to better describe how HPV38 resets NF-B to a more virus-advantageous setting, such as transcription of genes involved in the prevention of UVinduced apoptosis.

When NF-B signaling is inhibited by different strategies in 38E6E7 HFK, we have observed that inhibition of NF-B nuclear translocation had a marginal impact on cellular proliferation, while inhibition of IKK promoted a severe decrease of cellular growth. This observation highlights the existence of NF-B-independent pro-proliferation roles of IKK . Previous studies have also shown that IKK activity is not reduced to the unique function of I B phosphorylation and NF-B activation. For instance, IKK has been shown to target several other proteins such as FOXO3a, TSC1, Np73 , and p53, resulting in the promotion of cell survival and tumorigenesis (250). Further studies will be needed to better dissect the newly discovered IKK implication in other pathways than NF-B. It is also possible that the NF-Bindependent IKK -pro-proliferative roles in 38E6E7 HFK could be linked to its subcellular relocalization.

Interestingly, we observed that HPV38 E7 induces translocation of IKK in the nucleus where (i) it induces stabilization of Np73 and (ii) it is part of a negative regulatory complex containing Np73 , EZH2 and DNMT1. In the presented data, we have characterized the formation of this complex on several p53-regulated gene promoters with the exception of Survivin promoter that is, in contrast to the other investigated genes, an anti-apoptotic p53target gene. However it is important to explore a larger number of gene promoters that could be bound and regulated by the described protein complex. Np73 appeared to be the central component of this transcription inhibitory complex. Investigation of the variation gene expression upon dowregulation of Np73 expression in 38E6E7 HFK will help to identify the potential target genes and will provide insights on the function of the Np73 , IKK , EZH2, and DNMT1 complex.

The interaction of Np73 with EZH2 and DNMT1 and its correlation with histone H3Lys27me3 and CpG methylation, reveals a mechanism that can allow an efficient activity of Np73 in repressing gene transcription. This event shows that Np73 goes over the competitive action against p53, its binding can cause chromatin condensation and stable gene transcription silencing. EZH2 and other polycomb complex 2 components were previously reported to cooperate with DNMTs, including DNMT1, to form transcription inhibitory complexes. In keratinocytes expressing HPV38 E6 and E7, IKK -phosphorylated Np73 appears to highjack this protein complex in order to induce p53-regulated genes silencing. The formation of this multimeric protein complex in 38E6E7 HFK constitutes a mechanism that could also take place in other virus-immortalized cells and non-virus-related cancers. Actually, Np73 , EZH2, and DNMT1 are found accumulated in several cancers (251-254). In addition, IKK is also localized in the nucleus of numerous cancer cells lines (Results 3, Figure 4). These observations are in favor of a possible broader cooperation between IKK , Np73 , EZH2, DNMT1, and their not yet discovered other partners in the repression of p53-regulated proapoptotic genes during cellular transformation. A wider investigation of the gene promoters cobound by this protein complex will help to better describe their common action within the cells.

In addition, it remains of high interest to know if the formation of this complex can be a common event for some human cancers.

As cited above, although IKK is known to be localized in the cytoplasm, it is also accumulated in the nucleus of 38E6E7 HFK. This subcellular delocalization appears to be induced by the protein E7. However, the mechanism of IKK nuclear translocation is not yet known. As previously mentioned, the identification of E7 mutants that lose their ability to bind IKK will provide evidences on the possible implication of the E7/IKK interaction in the translocation of IKK in the nucleus.

A previous report has shown that IKK , in the nucleus of B-cell non-Hodgkin lymphoma, is part of a transcription regulation complex. In cooperation with BAFF-R, IKK phosphorylates histone H3 and leads to chromatin remodeling and gene transcription regulation (255). Thus, it is likely that IKK , in addition to Np73 , binds to a wider range of transcriptional regulatory factors in 38E6E7 HFK. One possible strategy to evaluate this possibility is to perform ChIPseq experiments using IKK antibodies. This experiment will lead to the identification of different promoters that are associated with IKK . The analysis cis responsive elements within these promoters will provide evidence whether IKK binds additional cellular transcription factors. Finally, ChIP and Re-ChIP experiments will be needed to validate the ChIP-seq results.

It is likely that such an approach will also result in the identification of novel and important genes that are regulated by IKK in transformed cells.

In summary, the presented data not only further clarify the HPV38 mechanisms in deregulating several cellular events, but also pave the way to novel studies aiming to elucidate the precise events that play a key role in cellular transformation.

Figure 1 .

 1 Figure 1. Electron micrograph of negatively stained papillomaviruses. Sources: left picture from Inserm unit 190, right picture from NIH-Visuals Online n AV-8610-3067.

Figure 2 .

 2 Figure 2. Papillomavirus phylogenetic tree based on the sequence of the L1 open reading frame.The numbers at the ends of each of the branches identify the Type; c-numbers refer to candidate types; all other abbreviations refer to animal papillomavirus types. The number at the inner semicircular symbols refer to the papillomavirus species and the outermost semicircular symbols identify papillomavirus genera. The tree was constructed using the MEGA5.1 program[START_REF] De Villiers | Cross-roads in the classification of papillomaviruses[END_REF].

  E1 and E2 proteins bind to the origin of replication and recruit host polymerases necessary for replication. Their activity ensures the maintenance of the genome at a low copy number in the basal cells (121). During mitosis, viral genomes are partitioned to the daughter cells through the binding of E2 to viral DNA and to protein complexes on host cell chromosomes (122-125). This observation was further investigated using bovine PV (BPV) E2 protein. At specific steps of cell cycle, BPV E2 interacts with the DNA helicase ChlR1, suggesting the involvement of ChlR1 in the loading of the viral genome to the host cell chromosomes during DNA amplification (126, 127).

Figure 3 .

 3 Figure 3. Human papillomavirus (HPV) genome and life cycle. Upper panel: Genomic organization of HPV16. Eight major open reading frames code for early (E) and late (L) genes. Locus control region (LCR) and the responsive element within are also shown. Lower panel: Schematic representation of HPV life cycle in cutaneous and mucosal epidermis. Infected cells are represented in red. They proliferate and migrate through the upper layers. HPV life cycle is tightly linked to keratinocytes differentiation (133).

  immunity and inflammation as well as modulation of apoptosis. Two other activating pathways included in the canonical one are not fully elucidated. The first is induced by DNA damage which activates casein kinase 2 (CK2). The second acts in response to phorbol esters, lipopolysaccharide (LPS), and cytokines stimuli, and involves the activation of IKKε and TANK-binding kinase (TBK). The other major NF-κB signaling is known as the alternative or non-canonical pathway. It is activated by several stimuli such as B-cell-activating factor, LT α/β, or CD40L, leading to the NF-κB-inducing kinase (NIK)-dependent activation of the homodimer IKKα. RelB and p100 form the NF-κB dimer which is activated by the degradation of the C-terminal region of p100 after phosphorylation by IKKα. This pathway is required for B cell maturation and survival, and formation of secondary lymphoid organs (187-189).

Figure 4 .

 4 Figure 4. Schematic representation of NF-κB signaling.NF-B canonical pathway is characterized by the p50/p65 NF-κB subunits which are inhibited by IκB. The phosphorylation of IκB by CK2, IKKε or IKK complex leads to its degradation. This pathway targets genes implicated in inflammation and apoptosis. The non-canonical NF-κB pathway involves the p100/RelB complex which is targeted by the IKKα homodimer. Phosphorylation of p100 causes its processing to the active form p52. The activation of this pathway is mainly responsible for the expression of genes involved in B cell development

  Studies on beta HPV types also led to the characterization of a novel mechanism involved in the regulation of intracellular levels of Np73 , an antagonist of the p53/p73-regulated pathways(234, 235). HPV38 E6 and E7 promote the accumulation of a specific form of p53 that positively regulates the transcription of Np73. In addition, HPV38 acts on a posttranslational level increasing Np73 half-life (235). HPV38 E6 and E7 expression in primary human keratinocytes promotes the translocation of I B-kinase-(IKK ) in the nucleus, which in turn associates with and phosphorylates Np73 at serine 422, leading to its stabilization and repression of several p53-regulated genes. Inhibition of IKK results in a rapid degradation of Np73 and a rescue of p53 function (235).

  is now well established that although HPV38 E6 and E7 fail to degrade p53, they possess efficient mechanisms to inhibit cellular cell cycle control and apoptosis induction (234). It has also been shown that human primary keratinocytes expressing HPV38 E6 and E7 gradually erode their telomeres (223). The shortening in telomere length correlates with genomic instability and chromosomal rearrangements that can result in the transformation of some cellular clones. The same study has shown that the transcriptional activation of hTERT expression can be at least partially inhibited by the knockdown of Np73 protein synthesis(223). This observation suggests the involvement of Np73 in the overexpression of hTERT in presence of HPV38 E6 and E7. Another study has investigated the status of hTERT expression in keratinocytes by comparing the effects of the expression E6 and E7 from different beta HPV types. E6 and E7 from HPV38 and HPV49 were the most significantly inducing hTERT expression in human keratinocytes(224). It was also reported that E6AP plays and important role in the beta HPV-mediated hTERT activation (242). E6 proteins from different beta HPV type interact with E6AP and NFX1-91 and induce telomerase activity (242).

Figure 1 .

 1 Figure 1. HPV38 E6 and E7 influence directly IKK and NF-B activatiesA/ Immunoblot on total proteins from cellular lysates of HFKs expressing E6 and E7 oncoproteins from HPV10, 14 or 38 or transfected with the pLXSN empty vector. Antibodies were used to look for the expression of phosphorylated-IkBa and phosphorylated-p65 compared to the total I B and p65 protein amount. B/In vitro kinase assay using same amounts of immunoprecipitated proteins from 38E6/E7HFKs expressing pRS38E6/E7 or with the empty pRS vector (left panel). IKK proteins were incubated with GST or GST-I B during 30min at 30°C in presence of [g-32P]ATP and radio activity is then detected. C/ HPV38 E6/E7HFKs expressing pRS38E6/E7 or pRS ampty were used to detecte by immunobloting the protein levels of phopho-I B and phospho-p65 compared to the total amounts of this proteins. D/ Quantitative PCR on the total cDNA obtained with RT-PCR on total RNA isolated from 38E6/E7HFKs expressing pRS38E6/E7 or containing the empty pRS vector. The expression levels of HPV38E6, HPV38E7, xIAP, cIAP1 and cIAP2 were analysed and normalized on the GAPDH expression level.

E7

  of IKK at serine 176 and IKK at serine 188 are hallmark of activation of the IKK complex. Higher amounts of phospho-

Figure 2

 2 Figure 2. NF-B pathway activation in primary keratinocytes expressing HPV38E6 or HPV38E7 Primary keratinocytes expressing individually the oncoproteins HPV38E6 and HPV38E7 or with a pLXSN empty vector were collected at early passages for immunobloting assay. Protein levels of phosphorylated IKK and phosphorylated I B were detected and compared to the total I B amounts and -actin.

Figure 3 .

 3 Figure 3. IKK interacts with HPV38 E7 and accumulated in the nucleusA/ Co-immunoprecipitation analysis using cell lysates from 293 cells transiently transfected with IKK Flag-38E7. Anti-IKK and anti-Flag antibodies were used for the immunoprecipitation and for immunoblotting. B/ Immunofluorescence staining of IKK and IKK in human forskin keratinocytes expressing HPV38E6 and E7 together. DAPI is used to detect the nuclei.

  transforming protein Tax constitutively activates NF-B, by physically

Chapter III: Discussion and conclusions

Alteration of p53 and NF-B pathways by E7 protein from cutaneous Human Papillomavirus type 38

Abstract

Viral infections contribute to 15-20% of all human cancers. Studying the mechanisms employed by the oncogenic viruses to induce cellular transformation is essential for a better understanding of the resulting cancers and the discovery of new mechanisms involved in cancer development which can be targeted in therapeutic approaches. Human papillomaviruses (HPVs) are small dsDNA viruses which have been clearly associated with certain cancers. They were first isolated from the skin of patients suffering from Epidermodysplasia Verruciformis (EV) having an increased susceptibility to infection by specific HPV types and to the development of non-melanoma skin cancer (NMSC). Certain cutaneous HPV types, such as 5, 8, and 38, are suspected to play a role in skin cancer development. However the direct role of cutaneous HPV in the etiology of cancer is still under debate. Previous studies from our laboratory have reported that HPV38 E6 and E7 proteins are able to immortalize human primary keratinocytes in vitro and in vivo. Cellular immortalization can be achieved through the deregulation of important signaling pathways including p53 and NF-B. In the present work, we have investigated the molecular mechanisms of p53 and NF-B pathways deregulation by E6 and E7 oncoproteins from HPV38 in human keratinocytes. We show here that HPV38 E6E7 induce the formation of a transcription repressor complex including IKK , Np73 , and polycomb group members EZH2 and DNMT1. The formation of this protein complex correlates with the inhibition of several p53-target genes, such as PIG3. We also report in these studies that HPV38 E6E7 activate NF-B pathway, which plays an important role in the survival of HPV38 E6E7-immortalized human keratinocytes upon TNF--and UVB-mediated apoptosis. In addition our data highlight E7 being the main HPV38 protein mediating p53 and NF-B deregulation. Our studies shed light on novel molecular mechanisms that could be important for HPV38-mediated cellular transformation.

Dérégulation des voies de signalisation p53 et NF-B par la protéine E7 du Papillomavirus Humain de type 38

Résumé

Les infections virales sont responsables de 15 à 20 % des cancers humains. L'étude des mécanismes moléculaires avec lesquels les virus oncogènes induisent la transformation cellulaire est essentielle pour la compréhension des cancers qui en résultent. Cela permettra également la découverte de nouveaux mécanismes pouvant être impliqués dans le développement de cancers, qui peuvent être ciblés par des approches thérapeutiques. Les virus du papillome humain (HPV) sont des petit virus à ADN qui futs isolés de la peau de patients souffrants de Epidermodysplasia Verruciformis (EV) qui cause un risque élevé d'infection par les HPV et le développement de cancer de la peau non mélanique (NMSC). Certains HPV cutanés, tels que HPV5, 8 et 38, sont suspectés de jouer un rôle dans de développement du cancer de la peau.