Human papillomavirus and the development of non-melanoma skin cancer.

Human papillomaviruses (HPV) are increasingly recognised as important human carcinogens. The best established association with human malignancy is that of high-risk mucosal HPV types and anogenital cancer. HPV-induced transformation of anogenital epithelia has been the subject of intense research which has identified the cellular tumour suppressor gene products, p53 and pRB, as important targets for the viral oncoproteins E6 and E7 respectively. Certain HPV types are also strongly associated with the development of non-melanoma skin cancer in the inherited disorder epidermodysplasia verruciformis (EV). However, in contrast with anogenital malignancy the oncogenic mechanisms of EV-HPV types remain uncertain, and there appears to be a crucial additional requirement for ultraviolet radiation. Cutaneous HPV types in the general population are predominantly associated with benign viral warts, but a role in non-melanoma skin cancer has recently been postulated. Polymerase chain reaction based HPV detection techniques have shown a high prevalence of HPV DNA, particularly in skin cancers from immunosuppressed patients and to a lesser extent in malignancies from otherwise immunocompetent individuals. No particular HPV type has yet emerged as predominant, and the role of HPV in cutaneous malignancy is unclear at present. It remains to be established whether HPV plays an active or purely a passenger role in the evolution of non-melanoma skin cancer.     

Comparative transforming potential of different human papillomaviruses associated with non-melanoma skin cancer

It is well established that high-risk human papillomaviruses (HPVs) that infect mucosal epithelia are the causative agents of cervical cancer. In contrast, the association of cutaneo-tropic HPV types with the development of non-melanoma skin cancer (NMSC) is less well defined. In this study, we have analysed the in vitro transforming potential of various cutaneous HPV types. Using oncogene cooperation assays with activated ras, we have shown that diverse cutaneous types, including 12, 14, 15, 24, 36 and 49, have significant transforming potential. Interestingly, most of this activity appears to be encoded by the E6 gene product. In contrast, the common HPV-10 exhibits no significant transforming potential in these assays. This difference may be a reflection of different patterns of cellular localization, with transforming E6s being nuclear and non-transforming being cytoplasmic. These results provide molecular support for a role of these viruses in the development of certain human malignancies.     

Molecular mechanisms of cervical carcinogenesis by high‐risk human papillomaviruses: novel functions of E6 and E7 oncoproteins

Over the last two decades, since the initial discovery of human papillomavirus (HPV) type 16 and 18 DNAs in cervical cancers by Dr. Harald zur Hausen (winner of the Nobel Prize in Physiology or Medicine, 2008), the HPVs have been well characterised as causative agents for cervical cancer. Viral DNA from a specific group of HPVs can be detected in at least 90% of all cervical cancers and two viral genes, E6 and E7, are invariably expressed in HPV‐positive cervical cancer cells. Their gene products are known to inactivate the major tumour suppressors, p53 and retinoblastoma protein (pRB), respectively. In addition, one function of E6 is to activate telomerase, and E6 and E7 cooperate to effectively immortalise human primary epithelial cells. Though expression of E6 and E7 is itself not sufficient for cancer development, it seems to be either directly or indirectly involved in every stage of multi‐step carcinogenesis. Epidemiological and biological studies suggest the potential efficacy of prophylactic vaccines to prevent genital HPV infection as an anti‐cancer strategy. However, given the widespread nature of HPV infection and unresolved issues about the duration and type specificity of the currently available HPV vaccines, it is crucial that molecular details of the natural history of HPV infection as well as the biological activities of the viral oncoproteins be elucidated in order to provide the basis for development of new therapeutic strategies against HPV‐associated malignancies. This review highlights novel functions of E6 and E7 as well as the molecular mechanisms of HPV‐induced carcinogenesis. Copyright © 2009 John Wiley & Sons, Ltd.     

Cellular steady-state levels of "high risk" but not "low risk" human papillomavirus (HPV) E6 proteins are increased by inhibition of proteasome-dependent degradation independent of their p53- and E6AP-binding capabilities

The group of mucosal epithelia-infecting human papillomaviruses (HPV) can be subdivided in "low" and "high risk" HPV types. Both types induce benign neoplasia (condyloma), but only the infection with a "high risk" HPV type is causally associated with an increased risk of developing anogenital tumors. The oncogenic potential of high risk HPVs resides at least partially in the viral E6 protein. The E6 protein targets the cellular p53 protein for proteasome-dependent degradation, which is associated with the immortalizing and transforming functions of these viruses. Recently the E6-dependent proteasome-mediated destabilization of additional cellular proteins (E6TP1, c-myc, Bak, hMCM7, human scribble, E6AP, MAGI-1) has been described, but the cellular mechanisms controlling the viral E6 protein stability itself have been so far not analyzed. In this study, we transiently expressed the E6 genes of the high risk HPV type 16, the low risk HPV types 6a and 11, and the cutaneous epithelia-infecting HPV types 5 and 8 from a eucaryotic expression vector and compared the cellular steady-state levels of the expressed E6 proteins. We demonstrated that the high risk HPV 16 E6 protein possesses the lowest steady-state level in comparison to the low risk HPV type E6 proteins and the cutaneous epithelia-infecting HPV type E6 proteins. Inhibition of cellular proteasome-dependent protein degradation led to an increase in steady-state levels of high risk but not of low risk E6 proteins. Analysis of functionally deficient HPV 16 E6 proteins in p53 null- and p53 wild-type-expressing cell lines revealed that the cellular steady-state level of this protein is influenced neither by its p53- nor its E6AP-binding abilities.     

Cellular transformation by human papillomaviruses: lessons learned by comparing high- and low-risk viruses

The oncogenic potential of papillomaviruses (PVs) has been appreciated since the 1930s yet the mechanisms of virally-mediated cellular transformation are still being revealed. Reasons for this include: a) the oncoproteins are multifunctional, b) there is an ever-growing list of cellular interacting proteins, c) more than one cellular protein may bind to a given region of the oncoprotein, and d) there is only limited information on the proteins encoded by the corresponding non-oncogenic PVs. The perspective of this review will be to contrast the activities of the viral E6 and E7 proteins encoded by the oncogenic human PVs (termed high-risk HPVs) to those encoded by their non-oncogenic counterparts (termed low-risk HPVs) in an attempt to sort out viral life cycle-related functions from oncogenic functions. The review will emphasize lessons learned from the cell culture studies of the HPVs causing mucosal/genital tract cancers.     

Search for cellular partners of human papillomavirus type 16 E2 protein

Human papillomaviruses (HPVs) are small, double-stranded DNA viruses that infect cutaneous and mucosal epithelia. Type 16 (HPV16) displays tropism to genital epithelia, giving rise to genital warts and cervical intraepithelial neoplasia (CIN), which is a precursor lesion to invasive carcinoma of the cervix. The great majority of human cervical cancers contain integrated HPV DNA where the E2 gene is usually disrupted, suggesting that the loss of the E2 protein is an important step in HPV-induced carcinogenesis. The HPV16 E2 protein is a regulatory protein that seems to be essential for creating favourable conditions for establishment of infection and proper completion of the viral life cycle. Recently, diverse activities of the E2 proteins have been described, but the molecular basis of these processes has not beenfully elucidated. Using a yeast two-hybrid system, we have identified epithelial cellular proteins that bind to the E2 protein of HPV16.     

Alpha- and betapapillomavirus E6/E7 genes differentially modulate pro-inflammatory gene expression

Keratinocytes, the target cell of human papillomavirus (HPV) infection, can produce numerous cytokines and pro-inflammatory molecules which are important for the generation of an effective immune response. How this biological response, which involves the tumor stroma, is affected by the HPV oncoproteins within the epithelial cell itself is not clear. Here it is shown that oncoproteins of different HPV genotypes (alpha- versus beta-HPV genus) alter the expression of pro-inflammatory molecules in early passage primary human keratinocytes and the immortalized cell line HaCaT. HPV5 E6/E7 oncoproteins significantly induced interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) expression. By contrast, the same molecules were down-regulated or not modulated in HPV16 E6/E7 transduced keratinocytes. Interestingly, HPV38 oncoproteins expression resulted in a lower induction of pro-inflammatory molecules, resembling the behavior displayed by the mucosal carcinogenic HPV16. Finally, inducible nitric oxide synthase (iNOS) expression levels and nitric oxide (NO) production were induced at similar levels by all the HPV genotypes tested. These results further emphasize the different biological activities among HPV genotypes, and offer new insights into HPV-associated skin diseases.     

Review: antibodies to cutaneous human papillomaviruses

Human papillomaviruses (HPVs) are small double-stranded DNA viruses that infect the epithelia of skin or mucosa. Cutaneous HPV is ubiquitous and many children have skin warts at some stage during childhood and a high proportion of the adult population have asymptomatic skin HPV infections. While a subset of mucosal HPV types are involved in cervical cancer, the role for cutaneous HPV types in skin cancer is still under debate. There is some evidence that the skin HPV types have mechanisms to induce skin cancer, but the role of HPV infection has never been proved in larger epidemiological studies and is not well understood. Cutaneous HPV serology has been employed more commonly during the last decade to help find a link between cutaneous HPV and skin cancer. This review covers the findings from cutaneous HPV serology studies published during the last decade and discusses differences in seroprevalence and risk factors.     

Human papillomavirus immortalization and transformation functions

The high risk HPVs (such as HPV-16 and HPV-18) that are associated with specific anogenital cancers encode two oncoproteins E6 and E7, which are expressed in the HPV positive cancers. The E7 protein functions in cellular transformation, at least in part, through interactions with pRB and the other pRB related 'pocket proteins'. The major target of the E6 oncoprotein encoded by the genital tract, cancer associated human papillomaviruses is p53. Several lines of evidence suggest that E6 and E7 have additional targets important to the oncogenic potential of the virus. Work from a number of laboratories has focused on determining other activities of HPV relevant to carcinogenesis and identifying additional cellular targets of E6 and E7. This paper will review the state of the field at the time of the 19th International Papillomavirus Workshop in September 2001 with respect to the HPV encoded oncoproteins.     

Human papillomaviruses: a growing field

A combination of functional studies on human papillomavirus (HPV) oncoproteins and epidemiological studies on persistence of HPV infection firmly established a role for HPV in the etiology of cervical cancers. Understanding the viral life cycle of HPVs has been more difficult. In this issue of Genes & Development, Wang et al. (pp. 181 – 194) describe an efficient method to propagate infectious HPV in differentiating epithelium, providing clear evidence for temporal separation of viral and cellular replication.     

Expression of HPV16 E5 produces enlarged nuclei and polyploidy through endoreplication

Anogenital cancers and head and neck cancers are causally associated with infection by high-risk human papillomavirus (HPV). The mechanism by which high-risk HPVs contribute to oncogenesis is poorly understood. HPV16 encodes three genes (HPV16 E5, E6, and E7) that can transform cells when expressed independently. HPV16 E6 and E7 have well-described roles causing genomic instability and unregulated cell cycle progression. The role of HPV16 E5 in cell transformation remains to be elucidated. Expression of HPV16 E5 results in enlarged, polyploid nuclei that are dependent on the level and duration of HPV16 E5 expression. Live cell imaging data indicate that these changes do not arise from cell-cell fusion or failed cytokinesis. The increase in nuclear size is a continual process that requires DNA synthesis. We conclude that HPV16 E5 produces polyploid cells by endoreplication. These findings provide insight into how HPV16 E5 can contribute to cell transformation.     

Koilocytosis: a cooperative interaction between the human papillomavirus E5 and E6 oncoproteins

A long-recognized, pathognomonic feature of human papillomavirus (HPV) infection is the appearance of halo or koilocytotic cells in the differentiated layers of the squamous epithelium. These koilocytes are squamous epithelial cells that contain an acentric, hyperchromatic nucleus that is displaced by a large perinuclear vacuole. However, the genesis of the cytoplasmic vacuole has remained unclear, particularly because both HPV DNA replication and virion assembly occur exclusively in the nucleus. In clinical biopsies, koilocytosis is observed in both low- and high-risk HPV infections; therefore, in this study, we demonstrated that the E5 and E6 proteins from both low- and high-risk HPVs cooperate to induce koilocyte formation in human cervical cells in vitro, using both stable and transient assays. Both E5 and E6 also induce koilocytosis in human foreskin keratinocytes but not in primate COS cells. Deletion of the 20 C-terminal amino acids of E5 completely abrogates koilocytosis, whereas a 10-amino acid-deletion mutant retains ~50% of its activity. Because the E6 protein from both the low- and high-risk HPVs is capable of potentiating koilocytosis with E5, it is apparent that the targeting of both p53 and PDZ proteins by E6 is not involved. Our data suggest new, cooperative functions for both the E5 and E6 proteins, hinting at additional targets and roles for these oncoproteins in the viral life cycle.     

Human papillomaviruses and non-melanoma skin cancer: basic virology and clinical manifestations

Human papillomaviruses (HPV) infect cutaneous and mucosal epithelia and induce benign and malignant lesions. Non-melanoma skin cancer (NMSC), encompassing basal cell carcinoma and squamous cell carcinoma (SCC), is the most frequent cancer in the Caucasian population, and the incidence has increased dramatically worldwide. Ultraviolet (UV) radiation is a major risk factor for NMSC, and cutaneous HPV is also considered to play an active role during the pathogenesis of these cancers. The first evidence for the involvement of HPV in NMSC was reported in patients with Epidermodysplasia verruciformis (EV). HPV types detected in skin tumours of these patients are referred to as EV/cutaneous HPV types belonging to the beta- and gamma-papillomaviruses (PV). Epidemiological studies have shown a higher risk of several EV/cutaneous HPV types for NMSC. Furthermore, in vitro and animal models show transforming properties of some PV types. The anti-apoptotic activities, and the delay of DNA repair mechanism caused by some EV/cutaneous HPV E6 proteins in response to UV-induced mutations, may lead to the persistence of DNA-damaged keratinocytes. Thus, specific EV/cutaneous HPV types as co-factors in association with UV-radiation and the immune system seem to be involved in the early pathogenesis of cutaneous SCC.     

Binding of PDZ proteins to HPV E6 proteins does neither correlate with epidemiological risk classification nor with the immortalization of foreskin keratinocytes

There is compelling evidence that high-risk human papillomaviruses (HPV) can cause cervical cancer. Strikingly, HPV16 and 18 account for ∼ 70% of all cervical cancers, whereas phylogenetically related types are found at much lower frequencies. Most likely, differences in the activities of the viral E6 and E7 oncoproteins account for the in vivo carcinogenicity. We demonstrate here that E6 proteins from low-risk HPV70 and possibly high-risk HPV82 interact and degrade PDZ proteins hDlg and Magi1 identical to HPV16E6 and HPV18E6. In contrast high-risk HPV66E6 did not bind or degrade hDlg or Magi1. We also show that low-risk HPV70 E6/E7 immortalizes normal human keratinocytes. Together with our previous analysis concerning p53 degradation, this shows that neither binding of E6 to p53, to E6AP, to Magi1 and hDlg, the degradation of hDlg and Magi1, nor immortalization of normal human keratinocytes seems to be a reliable predictor for carcinogenic behavior of HPV in the cervix.     

Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous cancers from renal transplant recipients.

The epidermodysplasia verruciformis (EV)-associated human papillomaviruses (HPVs) constitute a group of HPV genotypes isolated mostly from the cutaneous lesions of patients with the genetic disorder of EV. Broad-spectrum detection of EV HPVs in cutaneous lesions of non-EV patients was previously difficult because no EV HPV consensus PCR was available. We describe a nested PCR that enables the detection of all known EV HPV types at relatively low-copy-number levels. The deduced sequences of a 92-amino-acid stretch of the L1 open reading frames of all types are shown for convenient typing. The technique proved very valuable in viral studies of skin cancers from renal transplant recipients. A high prevalence (81%) of EV HPV types was found in skin cancer biopsies. A wide spectrum of EV HPV types that differed from HPV-5 and -8 was found to be involved. The technique also proved useful in detecting potentially novel EV HPV types in skin cancers. The relationship of these new types to known HPV types is demonstrated by phylogenetic tree analysis.     

The human papillomavirus E7 oncoprotein can uncouple cellular differentiation and proliferation in human keratinocytes by abrogating p21Cip1-mediated inhibition of cdk2

The high risk human papillomaviruses (HPVs) are associated etiologically with the majority of human cervical carcinomas. These HPVs encode two viral oncoproteins, E6 and E7, which are expressed consistently in cervical cancers. The function of these viral oncoproteins during a productive infection is to ensure viral replication in cells that have normally withdrawn from the cell division cycle and are committed to terminal differentiation. Expression of the E7 oncoprotein has been shown to lead to the abrogation of various negative growth regulatory signals, including a p53-mediated G₁ growth arrest, TGFβ-mediated growth inhibition, and quiescence of suprabasal keratinocytes. Here we describe a novel mechanism by which E7 can uncouple cellular proliferation and differentiation. In contrast to normal, differentiating keratinocytes, HPV-16 E7-expressing keratinocytes show delayed cellular differentiation and elevated cdk2 kinase activity despite high levels of p21^Cip1 and association of p21^Cip1 with cdk2. We show that the HPV E7 protein can interact with p21^Cip1 and abrogate p21^Cip1-mediated inhibition of cyclin A and E-associated kinase activities. Based on these findings, we propose that this capacity of the HPV E7 oncoprotein to overcome p21^Cip1-mediated inhibition of cdk2 activity during keratinocyte differentiation contributes to the ability of E7 to allow for cellular DNA synthesis in differentiated keratinocytes.     

Correlation between the presence of high-risk human papillomaviruses and Id gene expression in Syrian women with cervical cancer

Infection by high-risk human papillomaviruses (HPVs) is considered to be the central cause of invasive cervical cancer. Previously reported studies have shown that Id genes regulate cell invasion and metastasis in several human carcinomas including cervical cancer. In order to investigate the correlation between high-risk HPVs and Id genes in human cervical cancer, the presence of high-risk HPVs and their association with Id gene expression was examined using PCR methods and tissue microarray analyses in a cohort of 44 cervical cancer patients from Syria. This study showed that high-risk HPVs were present in 42 samples (95%) that represent invasive cervical cancers and that the most frequent high-risk HPV types in Syrian women were 33, 16, 18, 45, 52, 58, 35, 51 and 31. Furthermore, the expression of E6 oncoprotein of high-risk HPVs was found to correlate with overexpression of Id-1, but not of Id-2, Id-3 or Id-4 in the majority of invasive cervical cancer tissue samples. These data suggest that high-risk HPVs can enhance the progression of human cervical cancer through Id-1 regulation.