Human papillomavirus: E6 and E7 oncogenes

The International Journal of Biochemistry & Cell Biology 39 (2007) 2006–2011 Pathogens in focus Human papillomavirus: E6 and E7 oncogenes Ga¨elle Bo...
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The International Journal of Biochemistry & Cell Biology 39 (2007) 2006–2011

Pathogens in focus

Human papillomavirus: E6 and E7 oncogenes Ga¨elle Boulet a,1 , Caroline Horvath a,∗,1 , Davy Vanden Broeck b , Shaira Sahebali a , Johannes Bogers a a

AMBIOR, Laboratory for Cell Biology & Histology, University of Antwerp, Groenenborgerlaan 171, BE-2020 Antwerp, Belgium b International Centre for Reproductive Health (ICRH), Ghent University, De Pintelaan 185, 9000 Ghent, Belgium Received 8 March 2007; received in revised form 6 July 2007; accepted 6 July 2007 Available online 19 July 2007

Abstract The recognition of a causal relationship between human papillomaviruses and cancer almost 30 years ago led to a rapid expansion of knowledge in the field, resulting in the description of the main mediators of HPV-induced carcinogenesis, the viral proteins E6 and E7. These oncoproteins show a remarkable pleiotropism in binding host-cell proteins, with the tumour suppressor genes p53 and pRb as their major targets. These interactions induce proliferation, immortalization and malignant transformation of infected cells. The link between HPV and cervical cancer led to the development of molecular methods, often based on the detection of E6 and E7, for screening and diagnosis. Therapeutic vaccines and gene therapy are primarily directed at E6 and E7. Although prophylactic vaccines are available, further understanding of the viral life cycle and the mechanisms underlying HPV-induced oncogenesis is necessary to face the many challenges in the field of HPV and cancer. © 2007 Elsevier Ltd. All rights reserved. Keywords: Human papillomavirus; E6 and E7 oncoproteins; Cancer

1. Introduction Human papillomaviruses (HPVs) are small circular double-stranded DNA viruses that belong to the Papovaviridae family. The causal role of HPV in cancers of the uterine cervix has been firmly established biologically and epidemiologically. Most cancers of the vagina and anus are equally caused by HPV, as are a fraction of cancers of the vulva, penis and oropharynx. Other malignancies causally linked to HPV are non-melanoma skin cancer and cancer of the conjunctiva (Mu˜noz, Castellsagu´e, Berrington de Gonz´alez, & Gissman, 2006). ∗ 1

Corresponding author. Tel.: +32 3 265 3300; fax: +32 3 265 3301. E-mail address: [email protected] (C. Horvath). Both the authors contributed equally to this work.

1357-2725/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2007.07.004

Papillomaviruses were first described in 1907 by Giuseppe Ciuffo, but HPV remained largely unstudied until the advent of molecular virology in the 1970s (Ciuffo, 1907; Meisels & Fortin, 1976; Zur Hausen, 1976). Of the more than 100 different HPV types identified, 40 are known to infect the genital tract (Woodman, Collins, & Young, 2007). These mucosal HPV types are classified as “low-risk” and “high-risk” types based on the prevalence ratio in cervical cancer and its precursors. Low-risk HPV types, such as 6 and 11, induce benign lesions with minimum risk of progression to malignancy. By contrast, high-risk HPVs have higher oncogenic potential (Fehrmann & Laimins, 2003). Approximately 99% of cervical cancers contain HPV DNA of highrisk types, with type HPV16 being the most prevalent, followed by types 18, 31, 33 and 45. Cervical HPV infection is one of the most common sexually transmitted

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infections (Walboomers et al., 1999; Woodman et al., 2007). The discovery of the relationship between persistent high-risk HPV infection and cervical cancer initiated a rapid expansion of the field, which resulted in the description of the function of HPV E6 and E7 oncogenes in cervical carcinogenesis (Woodman et al., 2007). 2. Overview of pathogenesis The HPV genome is approximately 8000 bp in length and encodes eight open reading frames (ORFs), which are transcribed as polycistronic mRNAs (Hebner & Laimins, 2006). Regulatory sequences required for viral replication and transcription are concentrated in a non-coding region termed “upstream regulatory region” (URR) or “long control region” (LCR). The gene products can be divided into “early” (E) and “late” (L) proteins depending on the time of expression during the viral life cycle (Fehrmann & Laimins, 2003). The properties of these proteins are summarized in Table 1. HPV is perfectly adapted to its natural host tissue, the differentiating epithelial cells of skin or mucosae. Its life cycle is directly linked to epithelial cell differentiation and initiated when infectious particles enter cells in the basal layer. Following entry, HPV genomes are established as episomes, which replicate in synchrony with the cellular DNA. Following cell division, daughter cells migrate towards the suprabasal compartment, where uninfected keratinocytes initiate terminal differentiation, while HPV-infected cells enter the S-phase of the cell cycle resulting in amplification of viral replicates (Mu˜noz et al., 2006). The subsequent release of infectious virions in the environment is facilitated by disintegration of the epithelial cells occurring as a result of natural turnover (Fehrmann & Laimins, 2003). The critical molecules in viral replication are E6 and E7, which functionally inactivate the products of

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two important tumour suppressor genes, p53 and pRb, respectively. Both oncoproteins induce proliferation, immortalization and malignant transformation of the infected cells. The differences between the E6 and E7 proteins of high- and low-risk HPV types seem often of a more quantitative rather than a qualitative nature (Mu˜noz et al., 2006). E6 acts as repressor of apoptosis and mediates survival of severely damaged cells, while E7 functions as promoter for replication and cell growth. Both can independently immortalize human cells, but their joint function gives rise to an interesting complementary and synergistic effect, inducing a marked increase in transforming activity (Fig. 1) (Zur Hausen, 2000). The physical state of HPV DNA within the cell has been shown to predict the pathological course of HPV infections. An important difference between high- and low-risk HPV types is integration in the host genome in high-grade lesions and cancer (Scheurer, Tortolero-Luna, & Adler-Storthz, 2005). High-risk HPV types show more tendency towards genomic integration, whereas low-risk types are preferentially maintained as extrachromosomal circular episomes (Arends, Buckley, & Wells, 1998). Integration occurs downstream of the early genes E6 and E7, often in the E2 region. This results in an increased E6 and E7 expression due to loss of negative feedback control by the viral regulatory E2 protein. Moreover, integrant-derived transcripts are more stable than those originating from episomal viral DNA. The subsequent increase in transforming E6 and E7 proteins results in a growth advantage for cells with integrants over cells with episomal viral genes (Woodman et al., 2007). 3. Structure of E6 and E7 The E6 and E7 proteins of high-risk types are the main mediators of carcinogenesis due to their interactions with various cellular targets. E6 is one of the first

Table 1 Overview of HPV gene products Gene product

Description

E1 E2 E4 E5 E6 E7

Helicase function; essential for viral replication and control of gene transcription Viral transcription factor; essential for viral replication and control of gene transcription Interaction with cytoskeleton proteins; viral assembly Growth stimulation by interaction with growth factor receptors; downregulation surface HLA class I molecules Cell immortalization; p53-degradation; telomerase activation; anti-apoptotic effect; induction of genomic instability Cell immortalization; interaction with pRb and pRb-associated pocket proteins; transactivation of E2F-dependent promoters; induction of genomic instability Major capsid protein Minor capsid protein; role in recruiting viral genomes for encapsidation; involvement in nuclear transport of viral DNA

L1 L2 (E) early and (L) late.

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Fig. 1. Cellular interactions of E6 and E7 oncoproteins and their synergy in induction of cell immortalization. E6 activates telomerase and SRC kinases, and inhibits p53 and BAK. E7 inhibits pRb, with consequent release of E2F and upregulation of p16, which is inactivated by E7. In addition, E7 stimulates cyclins A and E, inactivates CKIs p21 and p27 and induces centriole amplification. E6 and E7 synergize in cell immortalization (dotted lines); E6 prevents apoptosis induced by high E2F levels, while E7 shields E6 from inhibition by p16.

genes expressed during HPV infection (Fehrmann & Laimins, 2003). The E6 ORF encodes a small protein of approximately 150 amino acids with a molecular weight of 16–18 kD. E6 proteins contain four C-x-x-C motifs, which are important in functions, such as transcriptional activation, transformation, immortalization and association with cellular proteins. In the case of high-risk mucosal HPV types, the E6 COOH-terminal region contains a PSD-95/Dlg/ZO1 (PDZ) domain, involved in the interaction with several PDZ domain-containing proteins (Fig. 2A) (Hebner & Laimins, 2006). The E7 oncogene encodes a low molecular weight protein of approximately 100 amino acids. E7 proteins incorporate three conserved regions (CR), the NH2 -terminal CR1 domain, the CR2 region and the COOH-terminal CR3 domain (Fig. 2B). The NH2 -terminal CR1 domain is necessary for cellular transformation and pRb degradation, but does not directly contribute to pRb binding. The CR2 region contains a conserved pRb-binding core sequence LxCxE and a casein kinase II phosphorylation site (CKII). The COOH-terminal domain consists of two C-x-x-C motifs separated by a 29/30-amino acid spacer. This region is implicated in the association of pRb and other host cellular proteins, in metal binding and may function as a dimerization domain (Hebner & Laimins, 2006; M¨unger et al., 2004).

Fig. 2. Structure of E6 and E7 oncoproteins. (A) Schematic representation of the E6 oncoprotein. The protein of approximately 150 amino acids contains four C-x-x-C motifs (blue), which are important in functions, such as transcriptional activation, transformation, immortalization and association with cellular proteins. The E6 COOH-terminus contains a PDZ-binding motif (green) involved in the interaction with several PDZ domain-containing proteins. (B) Schematic representation of the E7 oncoprotein. This predominantly nuclear polypeptide of approximately 100 amino acids incorporates three conserved regions (CR). The COOH-terminal CR3 domain contains two copies of the C-x-x-C motif related to the E6 sequences (blue). This region is implicated in association of pRb and other host cellular proteins, in metal binding and may function as a dimerization domain. The CR2 domain (red) contains the pRb-binding core sequence LxCxE and a phosphorylation site for casein kinase II (CKII). The NH2 -terminal CR1 domain is necessary for cellular transformation and pRb degradation, but does not directly contribute to pRb-binding (light blue).

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4. Biological function

Table 2 Cellular binding partners for E6

4.1. High-risk HPV E6 protein

Binding partner

Consequence of interaction

Bak Bax CBP/p300

Anti-apoptotic effect Anti-apoptotic effect Downregulation of p53-dependent transcription

c-myc

Prevention of myc-induced apoptosis Increase hTert transcription and telomerase activity

E6AP

Deregulation signal transduction in proliferating cells Essential factor E6 degradation actions

E6TP1

Inhibition Rap-mediated signalling

E6BP

Inhibition of terminal cell differentiation p53-Independent anti-apoptotic effect

hDlg

Deregulation of cell cycle Loss of cell differentiation

hScrib IRF-3 MAGI-1/2/3 Mcm7 Paxillin

Influence on cell adhesion and polarity Decrease of interferon-␤ transcription p53-Independent anti-apoptotic effect Overriding early G1-phase arrest point Disruption of actin cytoskeleton and cell matrix interactions

p53

Deregulation of cell cycle Anti-apoptotic effect

XRCC1

Interference with DNA repair efficiency

The most important function of high-risk E6 is binding of the tumour suppressor p53, a DNA-binding protein expressed in response to DNA damage or unscheduled induction of DNA replication, resulting in cell cycle arrest or apoptosis. Since HPV depends on the cellular DNA synthesis machinery and must stimulate S-phase progression for the replication of its genome, overexpression of p53 inhibits viral replication (Fehrmann & Laimins, 2003). E6 binds the p53 protein through a cellular ubiquitin-ligase, the E6-associated protein (E6-AP), which recruits the ubiquitin complex of enzymes, ubiquitinating lysines on p53 and initiating its proteolysis. Degradation of p53 bypasses the normal growth arrest signals at the G1/S and G2/M checkpoints and is the prime cause of chromosomal instability, with mutational consequences for HPV-positive cells and enhancement of integration of foreign DNA into the host-cell genome (Thomas, Pim, & Banks, 1999). Furthermore, E6 oncoproteins inhibit degradation of SRC-family kinases by E6-AP, stimulating mitotic activity (Zur Hausen, 2002). As well as their effects on p53 protein, high-risk E6 proteins activate telomerase, an enzyme responsible for replicating telomeric DNA at the ends of chromosomes. In normal somatic cells, telomerase activity is absent and telomeres shorten through successive cell divisions, initiating the natural pathway that leads to senescence and cell death. E6 can upregulate telomerase activity by transcriptional activation of the human telomerase reverse transcriptase (hTert) gene, encoding the telomerase catalytic subunit (Fehrmann & Laimins, 2003). High-risk E6 proteins also interact with several PDZ domain-containing substrates, inducing their ubiquitinmediated degradation. These substrates are implicated in the control of cell proliferation, cell polarity and adhesion, which further supports that E6 proteins contribute to HPV-induced malignancy (Massimi, Gammoh, Thomas, & Banks, 2004). E6 shows a remarkable pleiotropism in binding other host-cell proteins, leading to substantial functional consequences for E6-expressing cells, although they are not fully understood at present (Table 2) (Fig. 1). 4.2. High-risk HPV E7 protein E7 proteins are primarily localized in the nucleus, where they associate with retinoblastoma gene product (pRb) to facilitate progression into the S-phase of the cell cycle (Zur Hausen, 2002). In normal cells, pRb

CBP, CREB-binding protein; E6-AP, E6-associated protein; E6TP1, E6-targeted protein; E6BP, E6-binding protein; hDlg, human homologue of the Drosophila disc large tumour-suppressor protein; hScrib, human homologue of the Drosophila Scribble tumour-suppressor protein; IRF3, interferon regulatory factor-3; MAGI, membrane associated guanylate kinase with inverted structure domain; Mcm7, minichromosome maintenance protein 7.

is hypophosphorylated in early G1 and bound to E2F transcription factors, forming complexes that function as transcriptional repressors. Upon phosphorylation the complexes dissociate, allowing E2F to act as a transcriptional activator. By associating with hypophosphorylated pRb, E7 prevents its binding with E2F, thereby promoting cell cycle progression. Additionally, the E7-induced ubiquitin-mediated pRb degradation appears to be essential in efficiently overcoming cell cycle arrest (Fehrmann & Laimins, 2003). Besides pRb, E7 interacts with two other members of the pRb family, p107 and p130, which also negatively regulate E2F transcription. In addition, high-risk E7 stimulates the S-phase genes cyclin E and cyclin A, interacts with cyclin-kinase complexes and abrogates the inhibitory activities of cyclin-dependent kinase inhibitors (CKIs), such as p21CIP-1/WAF-1 and p27KIP-1 . These interactions are a major factor in growth stimulation of HPV-infected cells, uncoupling cyclin-dependent kinase activity from

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Table 3 Cellular binding partners for E7 Binding partner

Consequence of interaction

AP1 family members

Abrogation of IRF-1 transcriptional activity Enhancement of c-myc-induced transcription Activation of cylins A/E Interference with G2/M cell cycle transition

c-myc Cyclin A/E complexes Histone H1 kinase IGFBP3

Anti-apoptotic effect Stimulation of cell proliferation

Mi2␤

Abrogation of IRF-1 transcriptional activity E2F release with subsequent activation of S-phase genes Deregulation of cell cycle Activation of genes for cell cycle progression Growth stimulation via deregulation of cell cycle Growth stimulation via deregulation of cell cycle Inhibition of interferon signal pathways pRb degradation via proteasome Interference with initiation of transcription

pRb pRb-associated pocket proteins (p107/p130) p21CIP-1/WAF-1 p27KIP-1 p48 Subunit4 ATPase TBP

AP1, activator protein 1; IRF-1, interferon regulatory factor-1; IGFBP3, insulin-growth factor-binding protein 3; TBP, TATA boxbinding protein.

CKIs and interfering with the ability of p53 to induce G1 growth arrest following DNA damage (Zur Hausen, 2000). Another consequence of high-risk E7 expression is the induction of genomic instability. By inducing centriole amplification, E7 also induces aneuploidy of the E7-expressing cell, which contributes to tumorigenesis (Zur Hausen, 2002). Table 3 shows a variety of other cellular proteins that associate with E7 (Fig. 1). E6 and E7 are independently able to immortalize human cells, but their combined expression leads to a complementary and synergistic effect, which in turn is responsible for increased transforming efficiency. Whereas CKI INK4A counteracts the functions of E6, E7 bypasses this inhibition by directly activating cyclins A and E. E6, in turn, prevents E7-induced apoptosis by degrading apoptosis-inducing proteins (Fehrmann & Laimins, 2003). 5. Clinical implications The identification of specific HPV types as causative agents of cervical cancer and its precursor lesions led

to the development of molecular methods in screening and diagnosis. Accurate diagnosis of HPV infection relies on the detection of viral nucleic acids (Molijn, Kleter, Quint, & van Doorn, 2005). Presence of high-risk HPV DNA identifies women at particular risk of progression to cervical cancer. Polymerase chain reaction (PCR) is a widely used, highly sensitive method to detect HPV DNA. One approach is based on consensus primers that target conserved regions within the L1/E1 region and can therefore amplify a broad spectrum of HPV types. However, type-specific PCRs with primers that bind to E6 and E7 are potentially more reliable because the L1/E1 region can be disrupted during viral integration and because the E6 and E7 nucleotide sequence exhibits less variation (Morris, 2005). Nevertheless, the high prevalence of transient infections makes viral detection an incomplete means of identifying women at risk. HPV load and integration status have been suggested as type-dependent risk markers for cervical neoplastic progression. Quantitative real-time PCR methods targeting E6 or E7 allow the accurate determination of viral load and screening for integration status based on the E2/E6 ratio (Woodman et al., 2007). Another application of the strong etiological relation between HPV and cervical cancer is the prevention or treatment of this disease and other HPV-associated malignancies by HPV vaccines (Mahdavi & Monk, 2005; Roden & Wu, 2006; Stanley, 2006). Prophylactic HPV vaccines prevent infections through induction of capsid-specific neutralizing antibodies. Since the main tumorigenic effects of high-risk HPVs have been attributed to E6 and E7, these oncoproteins are targets for therapeutic vaccines that are presently in development, administering E6/E7 either in live vectors, as peptides, protein or nucleic acid form, or in cell-based vaccines (Mahdavi & Monk, 2005). E6 and E7 are also the primary targets in the development of gene therapy for HPV infections (Shillitoe, 2006). Targeting E6 and E7 with antisense RNA was the original method for specific blocking of RNA translation, eliminating the malignant phenotype of cervical cancer cells. Afterwards, ribozymes were designed to digest E6 and E7 RNA, inhibiting the transformed phenotype or preventing cell immortalization. Smallinterfering RNAs (siRNA) induce degradation of their target molecule through the RNA interference pathway (McManus & Sharp, 2002). Silencing of E6 and E7 mRNA transcripts by siRNAs has been shown to induce apoptosis or senescence of HPV-immortalized cells. Other approaches to the prevention of gene expression have received less attention, and include blocking of E6 effects by specific binding to an RNA molecule,

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