The CXCL12/CXCR4 Signaling Pathway: A New Susceptibility Factor in Human Papillomavirus Pathogenesis.

The productive human papillomavirus (HPV) life cycle is tightly linked to the differentiation and cycling of keratinocytes. Deregulation of these processes and stimulation of cell proliferation by the action of viral oncoproteins and host cell factors underlies HPV-mediated carcinogenesis. Severe HPV infections characterize the wart, hypogammaglobulinemia, infection, and myelokathexis (WHIM) immunodeficiency syndrome, which is caused by gain-of-function mutations in the CXCR4 receptor for the CXCL12 chemokine, one of which is CXCR41013. We investigated whether CXCR41013 interferes in the HPV18 life cycle in epithelial organotypic cultures. Expression of CXCR41013 promoted stabilization of HPV oncoproteins, thus disturbing cell cycle progression and proliferation at the expense of the ordered expression of the viral genes required for virus production. Conversely, blocking CXCR41013 function restored virus production and limited HPV-induced carcinogenesis. Thus, CXCR4 and its potential activation by genetic alterations in the course of the carcinogenic process can be considered as an important host factor for HPV carcinogenesis.

HPV16-E2 induces prophase arrest and activates the cellular DNA damage response in vitro and in precursor lesions of cervical carcinoma.

Cervical intraepithelial neoplasia (CIN) is caused by human papillomavirus (HPV) infection and is the precursor to cervical carcinoma. The completion of the HPV productive life cycle depends on the expression of viral proteins which further determines the severity of the cervical neoplasia. Initiation of the viral productive replication requires expression of the E2 viral protein that cooperates with the E1 viral DNA helicase. A decrease in the viral DNA replication ability and increase in the severity of cervical neoplasia is accompanied by simultaneous elevated expression of E6 and E7 oncoproteins. Here we reveal a novel and important role for the HPV16-E2 protein in controlling host cell cycle during malignant transformation. We showed that cells expressing HPV16-E2 in vitro are arrested in prophase alongside activation of a sustained DDR signal. We uncovered evidence that HPV16-E2 protein is present in vivo in cells that express both mitotic and DDR signals specifically in CIN3 lesions, immediate precursors of cancer, suggesting that E2 may be one of the drivers of genomic instability and carcinogenesis in vivo.

Transcriptional control of late differentiation in human keratinocytes by TAp63 and Notch.

We previously showed that in cervical carcinoma cells, the TAp63ß isoform of the p63 transcription factor is negatively interfering with the carcinogenic pathways promoting anchorage-independent growth. In this study, we have defined the mechanisms underlying the effects of TAp63ß through a transcriptome analysis of human keratinocytes overexpressing this protein. TAp63ß modulated expression of 1203 genes (944 activated and 259 repressed; P-value <0.05), notably genes involved in epithelial development and keratinocyte differentiation. In comparison, while TAp63γ acts similarly to TAp63ß to transactivate a selected panel of target genes, other p63 isoforms, including ΔNp63α, which is highly expressed in keratinocytes, are inactive. Upon induction of differentiation of primary human keratinocytes, we observed endogenous expression of TAp63ß and γ isoforms, along with transcriptional activation of selected target genes. Intriguingly, our data also indicated that TAp63ß activates transcription of members of the Notch pathway, which is known to promote keratinocyte differentiation. By inhibiting and activating the Notch pathway, we revealed a subset of TAp63ß-activated genes that were co-dependent on Notch for their expression. Our work demonstrates that the shorter TAp63 isoforms (TAp63ß/γ) are specifically induced in human keratinocytes and cooperate with Notch signalling to activate transcription of late differentiation genes supporting their role as putative tumor suppressors in HPV-associated tumorigenesis.

Mapping of HPV transcripts in four human cervical lesions using RNAseq suggests quantitative rearrangements during carcinogenic progression.

Two classes of Human papillomaviruses (HPV) infect the anogenital track: high risk viruses that are associated with risk of cervical cancer and low risk types that drive development of benign lesions, such as condylomas. In the present study, we established quantitative transcriptional maps of the viral genome in clinical lesions associated with high risk HPV16 or low risk HPV6b. Marked qualitative and quantitative changes in the HPV16 transcriptome were associated with progression from low to high grade lesions. Specific transcripts encoding essential regulatory proteins such as E7, E2, E1^E4 and E5 were identified. We also identified intrinsic differences between the HPV6b-associated condyloma transcript map and that of the HPV16-associated low grade CIN specifically regarding promoter usage. Characterization and quantification of HPV transcripts in patient samples thus establish the impact of viral transcriptional regulation on the status of HPV-associated lesions and may therefore help in defining new biologically-relevant prognosis markers.

E2 proteins of high risk human papillomaviruses down-modulate STING and IFN-κ transcription in keratinocytes.

In the early stages of human papillomavirus (HPV) infection, the viral proteins elicit specific immune responses that can participate to regression of ano-genital lesions. HPV E6 protein for instance can reduce type I interferon (IFN) including IFN-κ that is involved in immune evasion and HPV persistence. To evaluate the role of E2 protein in innate immunity in HPV16-associated cervical lesions, genome-wide expression profiling of human primary keratinocytes (HPK) transduced by HPV16 E2 was investigated using microarrays and innate immunity associated genes were specifically analyzed. The analyses showed that the expression of 779 genes was modulated by HPV16E2 and 92 of them were genes associated with innate immunity. Notably IFN-κ and STING were suppressed in HPK expressing the E2 proteins of HPV16 or HPV18 and the trans-activation amino-terminal domain of E2 was involved in the suppressive effect. The relationship between STING, IFN-κ and interferon stimulated genes (ISGs) in HPK was confirmed by gene silencing and real time PCR. The expression of STING and IFN-κ were further determined in clinical specimens by real time PCR. STING and IFN-κ were down-modulated in HPV positive low grade squamous intraepithelial lesions compared with HPV negative controls. This study demonstrates that E2 proteins of high risk HPV reduce STING and IFN-κ transcription and its downstream target genes that might be an immune evasion mechanism involved in HPV persistence and cervical cancer development.

Genome-wide analysis of high risk human papillomavirus E2 proteins in human primary keratinocytes.

The E2 protein is expressed in the early stage of human papillomavirus (HPV) infection that is associated with cervical lesions. This protein plays important roles in regulation of viral replication and transcription. To characterize the role of E2 protein in modulation of cellular gene expression in HPV infected cells, genome-wide expression profiling of human primary keratinocytes (HPK) harboring HPV16 E2 and HPV18 E2 was investigated using microarray. The Principle Components Analysis (PCA) revealed that the expression data of HPV16 E2 and HPV18 E2-transduced HPKs were rather closely clustered. The Venn diagram of modulated genes showed an overlap of 10 common genes in HPV16 E2 expressing HPK and HPV18 E2 expressing HPK. These genes were expressed with significant difference by comparison with control cells. In addition, the distinct sets of modulated genes were detected 14 and 34 genes in HPV16 E2 and HPV18 E2 expressing HPKs, respectively.

Localization of HPV-18 E2 at mitochondrial membranes induces ROS release and modulates host cell metabolism.

Papillomavirus E2 proteins are predominantly retained in the nuclei of infected cells, but oncogenic (high-risk) HPV-18 and 16 E2 can shuttle between the host nucleus and cytoplasm. We show here that cytoplasmic HPV-18 E2 localizes to mitochondrial membranes, and independent mass spectrometry analyses of the E2 interactome revealed association to the inner mitochondrial membrane including components of the respiratory chain. Mitochondrial E2 association modifies the cristae morphology when analyzed by electron microscopy and increases production of mitochondrial ROS. This ROS release does not induce apoptosis, but instead correlates with stabilization of HIF-1α and increased glycolysis. These mitochondrial functions are not shared by the non-oncogenic (low-risk) HPV-6 E2 protein, suggesting that modification of cellular metabolism by high-risk HPV E2 proteins could play a role in carcinogenesis by inducing the Warburg effect.

Human papillomavirus proteins as prospective therapeutic targets.

Human papillomaviruses (HPV) are the causative agents of a subset of cervical cancers that are associated with persistent viral infection. The HPV genome is an ∼8 kb circle of double-stranded DNA that encodes eight viral proteins, among which the products of the E6 and E7 open reading frames are recognized as being the primary HPV oncogenes. E6 and E7 are expressed in pre-malignant lesions as well as in cervical cancers; hence these proteins have been extensively studied as potential targets for HPV therapies and novel vaccines. Here we review the expression and functions of E6 and E7 in the viral vegetative cycle and in oncogenesis. We also explore the expression and functions of other HPV proteins, including those with oncogenic properties, and discuss the potential of these molecules as alternative therapeutic targets.

HPV-18 E2

The Human Papillomavirus (HPV) E4 is known to be synthesized as an E1^E4 fusion resulting from splice donor and acceptor sites conserved across HPV types. Here we demonstrate the existence of 2 HPV-18 E2^E4 transcripts resulting from 2 splice donor sites in the 5' part of E2, while the splice acceptor site is the one used for E1^E4. Both E2^E4 transcripts are up-regulated by keratinocyte differentiation in vitro and can be detected in clinical samples containing low-grade HPV-18-positive cells from Pap smears. They give rise to two fusion proteins in vitro, E2^E4-S and E2^E4-L. Whereas we could not differentiate E2^E4-S from E1^E4 in vivo, E2^E4-L could be formally identified as a 23 kDa protein in raft cultures in which the corresponding transcript was also found, and in a biopsy from a patient with cervical intraepithelial neoplasia stage I-II (CINI-II) associated with HPV-18, demonstrating the physiological relevance of E2^E4 products.

Loss of HPV16 E2 Protein Expression Without Disruption of the E2 ORF Correlates with Carcinogenic Progression.

Integration of the viral DNA in the cellular genome has been suggested to be critical in carcinogenic progression of HPV-associated cervical neoplasia. This event can be accompanied by disruption of the open reading frame (ORF) encoding the E2 repressor, thus leading to transcriptional up-regulation of the E6 and E7 viral oncogenes. At this stage, it is unclear whether disruption of the E2 ORF is mandatory for carcinogenic progression. We measured E2 RNA and protein expression in clinical samples of various grades of HPV16-associated cervical neoplasia and compared it with the status of the viral genome. RNA extracted from paraffin embedded tissues was hybridized to specific probes and quantified by the NanoString technology. Protein expression was appreciated by immunohistochemistry and the status of viral DNA was determined by in situ hybridization, all performed on serial sections of the same samples. E2 protein was found highly expressed in CIN1, CIN2 lesions where the HPV DNA was highly replicative, while it was decreased in more advanced grade lesions where replication is decreased or lost (CIN3 and SCC). In contrast, E2 transcripts could be elevated even in conditions of no or low expression of the protein, as found in the Caski cell line. Our data demonstrate that integration of the viral DNA in the cellular genome does not always lead to disruption of the E2 ORF and drastic reduction of E2 transcripts, while in contrast, expression of the E2 protein is always drastically reduced.

The human papillomavirus E6 oncogene represses a cell adhesion pathway and disrupts focal adhesion through degradation of TAp63ß upon transformation.

Cervical carcinomas result from cellular transformation by the human papillomavirus (HPV) E6 and E7 oncogenes which are constitutively expressed in cancer cells. The E6 oncogene degrades p53 thereby modulating a large set of p53 target genes as shown previously in the cervical carcinoma cell line HeLa. Here we show that the TAp63ß isoform of the p63 transcription factor is also a target of E6. The p63 gene plays an essential role in skin homeostasis and is expressed as at least six isoforms. One of these isoforms, ΔNp63α, has been found overexpressed in squamous cell carcinomas and is shown here to be constitutively expressed in Caski cells associated with HPV16. We therefore explored the role of p63 in these cells by performing microarray analyses after repression of endogenous E6/E7 expression. Upon repression of the oncogenes, a large set of p53 target genes was found activated together with many p63 target genes related to cell adhesion. However, through siRNA silencing and ectopic expression of various p63 isoforms we demonstrated that TAp63ß is involved in activation of this cell adhesion pathway instead of the constitutively expressed ΔNp63α and ß. Furthermore, we showed in cotransfection experiments, combined with E6AP siRNA silencing, that E6 induces an accelerated degradation of TAp63ß although not through the E6AP ubiquitin ligase used for degradation of p53. Repression of E6 transcription also induces stabilization of endogenous TAp63ß in cervical carcinoma cells that lead to an increased concentration of focal adhesions at the cell surface. Consequently, TAp63ß is the only p63 isoform suppressed by E6 in cervical carcinoma as demonstrated previously for p53. Down-modulation of focal adhesions through disruption of TAp63ß therefore appears as a novel E6-dependent pathway in transformation. These findings identify a major physiological role for TAp63ß in anchorage independent growth that might represent a new critical pathway in human carcinogenesis.

Tumor suppressor or oncogene? A critical role of the human papillomavirus (HPV) E2 protein in cervical cancer progression.

The papillomavirus (PV) E2 proteins have been shown to exert many functions in the viral cycle including pivotal roles in transcriptional regulation and in viral DNA replication. Besides these historical roles, which rely on their aptitude to bind to specific DNA sequences, E2 has also been shown to modulate the host cells through direct protein interactions mainly through its amino terminal transactivation domain. We will describe here some of these new functions of E2 and their potential implication in the HPV-induced carcinogenesis. More particularly we will focus on E2-mediated modulation of the host cell cycle and consequences to cell transformation. In all, the HPV E2 proteins exhibit complex functions independent of transcription that can modulate the host cells in concert with the viral vegetative cycle and which could be involved in early carcinogenesis.

A pivotal role for CXCL12 signaling in HPV-mediated transformation of keratinocytes: clues to understanding HPV-pathogenesis in WHIM syndrome.

The WHIM syndrome, which features high susceptibility to human papillomavirus (HPV) infection, is a rare immunodeficiency associated with autosomal dominant heterozygous mutations of the CXCR4 chemokine receptor. CXCL12 and its receptors, CXCR4 and CXCR7, are linked to tumorigenesis, and we reported that abnormal expression of CXCL12 in epidermal keratinocytes correlates with HPV infection. However, the HPV-related pathologies observed in WHIM patients remain mechanistically unexplained. We show that keratinocytes immortalized by oncogenic HPV16 or HPV18 upregulate CXCL12 and its receptors in a manner dependent upon expression of the viral proteins E6 and E7. Autocrine signaling activated by CXCL12-engagement of its receptors controls motility and survival of the infected cells. Strikingly, expression of a WHIM syndrome-related gain-of-function CXCR4 mutant confers transforming capacity to HPV18-immortalized keratinocytes. These results establish a pivotal role for CXCL12 signaling in HPV-mediated transformation and provide a mechanistic basis for understanding HPV pathogenesis in WHIM syndrome.

HPV16 E2 is an immediate early marker of viral infection, preceding E7 expression in precursor structures of cervical carcinoma.

The viral E2 gene product plays a crucial role in the human papillomavirus (HPV) vegetative cycle by regulating both transcription and replication of the viral genome. E2 is a transcriptional repressor of the E6 and E7 viral oncogenes for HPV types 16 and 18, which are involved in cervical cancers. Using new polyclonal antibodies against the HPV16 E2 protein, we showed that E2 is expressed at various precursor stages of cervical carcinoma by immunohistochemistry on paraffin-embedded clinical samples. E2 was found to be highly expressed in the nuclei and cytoplasm of cells forming the intermediate and upper layers of cervical intraepithelial neoplasia (CIN). We could show that the expressions of E2 and p16(INK4a) (surrogate marker for oncogenic E7 expression) were exclusive in most of the cases, thus implying that E2 is not expressed together with high levels of E7. Moreover, we found that E2 is expressed in a subset of columnar cells adjacent to the CIN. We could show that expression of E2 is topologically distinct from the proliferation markers p63 and Ki67, whereas it coincides with the expression of cytokeratin K13, a marker of squamous cell differentiation. Expression of E2 also topologically coincides with episomal amplification of viral genomes in the upper layers of CIN1. These in vivo data thus validate previous assumptions of the crucial role of E2 in the early steps of HPV infection and of its negative link with expression of the viral E6 and E7 oncogenes.

The human papillomavirus type 18 E2 protein is a cell cycle-dependent target of the SCFSkp2 ubiquitin ligase.

The human papillomavirus type 18 (HPV-18) E2 gene is inactivated in cervical carcinoma after integration of the viral DNA into the host cellular genome. Since E2 represses the transcription of the two viral oncogenes E6 and E7, integration which allows their strong expression is considered a major step in transformation by HPV. We show here that E2 is specifically degraded at the end of the G(1) phase in a Brd4-independent manner, implying that its regulatory functions are cell cycle dependent. Degradation of E2 occurs via the Skp1/Cullin1/F-box Skp2 (SCF(Skp2)) ubiquitin ligase, since silencing of Skp2 induces stabilization of E2. In addition, the amino-terminal domain of E2 can interact with Skp2 as shown by coimmunoprecipitation experiments. We previously showed that E2 inhibits the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase, leading to accumulation of several of its substrates. We demonstrate here that Skp2, which is a known APC/C substrate in G(1), is also stabilized by E2. Therefore, by negative feedback, SCF(Skp2) activation could lead to E2 degradation and E6/E7 expression specifically in the late G(1) phase. Moreover, since the SCF(Skp2) can trigger S-phase entry and Skp2 itself is a known oncogene, we believe that E2-mediated accumulation of Skp2, together with E2 degradation leading to putative release of E6 and E7 inhibition, could induce premature S-phase entry in HPV-infected cells, pointing to a direct role of E2 in the early steps of HPV-mediated transformation.

Transcriptional regulation of the papillomavirus oncogenes by cellular and viral transcription factors in cervical carcinoma.

Human papillomaviruses (HPV) are small DNA viruses that contain a compact and non-redundant genome. HPV, with the help of only few genes, can achieve a complete vegetative cycle specifically in the epidermal and mucosal keratinocytes. Modification of the host cell transcriptional regulation is one of the major ways to regulate the viral production and maturation. The vegetative cycle of papillomaviruses is linked to terminal differentiation of the epithelium and is dependent on the host cell regulatory networks for transcriptional control. The mucosal high risk HPV16 and HPV18 types have been the main models to explore this transcriptional regulation mainly because they are prevalent in cervical cancer as the best studied virally induced cancers in human. In addition, the availability of cell lines, grown from cervical cancers containing integrated HPV16 or 18, represent versatile in vitro models for transcription studies. We will describe here some aspects of the transcriptional regulation that contribute to cell specificity, the basis of which is not yet fully understood despite efforts of numerous groups during the past two decades. Another specificity of small DNA viruses is the multifunctional characteristics of their regulatory proteins due to extreme genomic constraint. We will describe the role played by the viral E2 proteins in the transcriptional repression of the high risk HPV oncogenes and its implication in cervical cancer.

A new E6/P63 pathway, together with a strong E7/E2F mitotic pathway, modulates the transcriptome in cervical cancer cells.

Cervical carcinoma is associated with certain types of human papillomaviruses expressing the E6 and E7 oncogenes, which are involved in carcinogenesis through their interactions with the p53 and pRB pathways, respectively. A critical event on the path to malignant transformation is often manifested by the loss of expression of the viral E2 transcription factor due to the integration into the host genome of the viral DNA. Using microarrays, we have previously shown that reintroduction of a functional E2 in the HeLa cervical carcinoma cell line activates a cluster of p53 target genes while at the same time severely repressing a group of E2F target genes. In the present study, using new high-density microarrays containing more than 22,000 human cDNA sequences, we identified a novel p63 pathway among E2-activated genes and 38 new mitotic genes repressed by E2. We then sought to determine the pathways through which these genes were modulated and used an approach that relies on small interfering RNA to demonstrate that the p63 target genes were activated through silencing of the E6/E6AP pathway while the mitotic genes were mainly repressed through E7 silencing. Importantly, a subset of the mitotic genes was shown to be significantly induced in biopsies of stage IV cervical cancers, which points to a prominent E7 pathway in cervical carcinoma.

High-risk but not low-risk HPV E2 proteins bind to the APC activators Cdh1 and Cdc20 and cause genomic instability.

Human papillomaviruses (HPVs) from the high-risk group are associated with cervical cancer, in contrast to HPVs from the low-risk group which are associated with benign lesions of the genital tract. Here, we show that high-risk, but not low-risk HPV E2 proteins, promote a mitotic block, often followed by metaphase-specific apoptosis, and which is independent of the viral oncogenes E6 and E7. High-risk HPV E2-expressing cells also show polyploidy, chromosomal mis-segregation and centrosome amplification leading to genomic instability. We link these defects to a specific and unusually strong interaction between high-risk E2 and both Cdc20 and Cdh1, two activators of the Anaphase Promoting Complex (APC), abnormal localization of Cdh1, and accumulation of APC substrates like cyclin B, in vivo. The finding that high-risk, but not low-risk HPV E2 proteins, induce genomic instability, raises the intriguing possibility that E2 proteins play a role in the oncogenic potential of high-risk papillomaviruses.

Nucleo-cytoplasmic shuttling of high risk human Papillomavirus E2 proteins induces apoptosis.

Human Papillomavirus (HPV) E2 proteins are the major viral regulators of transcription and replication during the viral life cycle. In addition to these conserved functions, we show that E2 proteins from high risk HPV types 16 and 18, which are associated with cervical cancer, can induce apoptosis. In contrast, E2 proteins from low risk HPV types 6 and 11, which are associated with benign lesions, do not cause cell death. We show that the ability to induce apoptosis is linked to the intracellular localization of the respective E2 proteins rather than to inherent properties of the proteins. Although low risk HPV E2 proteins remain strictly nuclear, high risk HPV E2 proteins are present in both the nucleus and the cytoplasm of expressing cells due to exportin-1 receptor (CRM1)-dependent nucleo-cytoplasmic shuttling. Induction of apoptosis is caused by accumulation of E2 in the cytoplasm and involves caspase 8 activation. We speculate that disruption of the E2 gene during viral genome integration in cervical carcinoma provides a means to avoid E2-induced apoptosis and allow initiation of carcinogenesis.

Genomic organization of amplified MYC genes suggests distinct mechanisms of amplification in tumorigenesis.

Integration of the human papillomavirus (HPV) genome into the host genome is associated with the disruption of the HPV E2 gene and with amplification and rearrangement of the viral and flanking cellular sequences. Molecular characterization of the genomic structures of coamplified HPV sequences and oncogenes provides essential information concerning the mechanisms of amplification and their roles in carcinogenesis. Using fluorescent hybridization on stretched DNA molecules in two cervical cancer-derived cell lines, we have elucidated the genomic structures of amplified regions containing HPV/myc genes over several hundreds of kilobases. Direct visualization of hybridization signals on individual DNA molecules suggests that overreplication and breakage-fusion-bridge-type mechanisms are involved in the genomic instability associated with HPV cervical cancers. Further analysis from two other genital cancer-derived cell lines reveals a recurrent motif of amplification, probably generated by a common mechanism involving overreplication upon viral integration. Interestingly, different amplification patterns seem to be correlated with the disease outcome, thus providing new insights into HPV-related cancer development and tumor progression.