Human Papillomavirus Infections in Lung Cancer. Detection of E6 and E7 Transcripts and Review of the Literature
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ANTICANCER RESEARCH 27: 2697-2704 (2007) Short Review Human Papillomavirus Infections in Lung Cancer. Detection of E6 and E7 Transcripts and Review of the Literature LAURA GIULIANI1, CARTESIO FAVALLI1, KARI SYRJANEN2 and MARCO CIOTTI1 1Laboratory of Clinical Microbiology and Virology, University Hospital Tor Vergata, Rome, Italy; 2Department of Oncology and Radiotherapy, Turku University Hospital, Turku, Finland Abstract. Lung cancer is the leading cause of cancer related Lung cancer is the leading cause of cancer-related death death in Western countries. Several factors have been implicated in Western countries (8). Cigarette smoking, radiation, in its aetiology: cigarette smoking, environmental pollution, environmental pollution and exposure to asbestos are asbestos and genetic factors. The possible involvement of human considered the main risk factors for lung cancer. However, papillomavirus (HPV) in bronchial squamous cell lesions was they cannot explain all lung cancer cases and there is a first suggested in 1979 by Syrjänen. Since then, several studies possibility that additional risk factors can play a role in have confirmed the presence of HPV DNA in about 20% of bronchial carcinogenesis as suggested in the early 1980’s by lung cancer cases examined, with HPV16 and 18 as the two Syrjanen who described in bronchial carcinomas epithelial most frequently found oncogenic viral types. More recently, these changes resembling those typical of HPV-induced exophytic data have been supported by the detection of E6 and E7 and flat condylomas of the genital tract (9- 11). These transcripts in HPV-positive lung cancer cases, reinforcing the morphological studies were later confirmed by reports on hypothesis that oncogenic HPVs could act as cofactors in detection of HPV-DNA in bronchial carcinomas (11-15). bronchial carcinogenesis. This published literature is briefly In contrast other authors did not find HPV DNA in reviewed and new data of the authors on detection of E6 and lung tumors and failed to confirm such association (16- E7 transcripts in lung cancer samples are presented. 19). One of the reasons for these negative findings could be the different sensitivity of the techniques used or High risk human papillomaviruses (HPVs) are the prime inappropriate choice of primers. In fact, during the etiological agents for carcinoma of the uterine cervix. They integration of the virus into the host cell genome, part of are actually found in almost 100% of cervical cancer cases the L1 gene may be lost as well as the E2 gene and the (1, 2). It has been estimated that 15-20% of all human consensus primers MY09/MY11, frequently used to screen malignancies could be related to oncogenic HPVs (2-4). In these biopsies could then be inadequate for amplification. recent years, data have accumulated implicating the How does the virus get into this location? It has been involvement of HPV in squamous cell tumors at other speculated that HPV reaches the lungs via the bloodstream mucosal sites, such as the oral cavity, oesophagus, sino-nasal coming from distant infected sites such as the genital tract. tract, larynx and bronchi (2). The oncogenic power of HPV HPV DNA was detected in the blood of women with is due to its ability to interfere with the cell-cycle and cervical infection (20). In the light of the data reported tumour suppressive functions of the cell through its above, demonstration of E6 and E7 oncogenes and their oncoproteins E6 and E7, which inactivate p53 and Rb transcripts is crucial in order to elucidate the role of HPV in proteins, respectively (3-7). lung carcinogenesis. Lung Cancer Correspondence to: Dr. Marco Ciotti, Laboratory of Clinical Lung cancer is one of the leading causes of cancer-related Microbiology and Virology, University of Tor Vergata, Viale deaths worldwide. Cigarette smoking is responsible for Oxford, 81, 00133, Rome, Italy. Tel: +39 06 20902087, Fax: +39 06 20902078, e-mail: [email protected] about 90% of lung cancer cases. However, less than 20% of smokers develop lung cancer (21). Therefore, other Key Words: HPV infections, HPV-DNA, E6 and E7 transcripts, etiological agents, including genetic factors, functional lung cancer, short review. inactivation of tumor suppressor genes such as p53, Rb, p16, 0250-7005/2007 $2.00+.40 2697 ANTICANCER RESEARCH 27: 2697-2704 (2007) and infection with oncogenic types of HPV have been We recently studied the expression of E6 and E7 implicated in lung carcinogenesis (16, 22). Some reports oncogenes in six fresh lung tumour biopsies positive for have stressed an increased risk of developing lung cancer in HPV16 (80). To our knowledge, only one previous study women with anogenital malignancies (23, 24). An increased using in situ hybridization has addressed this issue in lung risk has been also observed in meat handlers (25-28). All cancer (34), despite the fact that transcriptional activity this epidemiological evidence supports a possible role for of E6 and E7 has been widely analysed in cervical HPV in lung carcinogenesis. carcinomas and in HPV transformed cell lines (82-87). In these studies, the transcription process of HPV appears Evidence for HPV Involvement to be quite complex. A common promoter (P97 in HPV16) controls the transcription of both E6 and E7 The accumulating data implicating a relationship between oncogenes using an alternative splicing mechanism. A HPV infection and lung carcinogenesis is derived from the common splice donor site at nt 226 and two different following approaches. splice acceptor sites at nts 409 and 526 give rise to three different transcripts. An unspliced E6/E7 transcript Morphological evidence. In 1979, Syrjanen reported encodes the E6 protein. The E7 protein in turn is morphological changes in squamous cell carcinomas of the encoded by the splicing products E6*I (acceptor site at lung closely resembling those seen in genital HPV lesions nucleotide 409) and E6*II (acceptor site at nucleotide (10, 11). These observations led to the hypothesis that HPV 526) (83). A novel promoter with a major start site could have a role also in bronchial carcinogenesis. around 670 nt (P670) in the E7 ORF was identified in an HPV16 transformed cell line derived from a vulvar Immunohistochemistry. Immunohistochemistry (IHC) has intraepithelial neoplasm (84). been used only sporadically to detect HPV antigens in lung In our study, the transcription pattern in lung tumors was cancer because it was soon replaced by the hybridisation deduced from three different PCR protocols carried out in assays in HPV detection. Hence, only two cases of HPV16-positive patients and in control CaSki cells (Figure squamous cell papillomas (SCPs) were analyzed by IHC as 1). Amplification with primers encompassing the E7 ORF reported by Trillo et al. (29). promptly showed the presence of E7 specific transcripts in five out of six patients as well as in CaSki cells (Figure 1, Detection of HPV DNA. Several different techniques have experiment A). been used to detect HPV-DNA in lung cancer cases: dot A more complex pattern was obtained when the E6 blot, Southern blot, ISH and PCR. These studies are transcription was investigated. The nested RT-PCR with summarised in Table I, (12-14, 29-81). The detection rate of primers encompassing the E6 ORF gave negative results in HPV DNA vary from 0% to 100% probably due either to four of these patients, while the remaining two showed a the different sensitivity of the techniques used and to 365-bp transcript (corresponding to the unspliced form of primers choice. Of all bronchial carcinomas analysed to E6) and a 183-bp transcript corresponding to the E6*I date, about 20% contain HPV DNA. HPV16 and 18 are the isoform, respectively (Figure 1, experiment B). The third most frequently found, while HPV6 and 11 are less RT-PCR protocol carried out with primers encompassing frequent. Multiple infections are sporadically described. the E6/E7 region showed a 166-bp transcript in all patients Among the high-risk group HPV31, 33 and 35 have also except two (Figure 1, experiment C). This band was also been found in scattered lesions. seen in CaSki cells together with two minor additional upper bands. Interestingly, the unspliced form (365-bp) of Detection of oncogenic transcripts. The possible role of E6 transcript was identified in only one tumor. It is oncogenic HPV types in lung carcinogenesis is suggested by important to emphasize that the synthesis of E6 protein is the presence of HPV DNA in bronchial carcinomas (Table mainly derived from a bicistronic mRNA, containing full- I). However, the presence of HPV DNA alone is not length E6 and E7 sequences (85). sufficient to support an active role of the virus in lung Taken together, our small study showed that all tumors carcinogenesis. Synthesis of E6 and E7 oncoproteins is a except one contained E6 and E7 RNA transcripts. The condition sine qua non for cell transformation and absence of viral transcripts in one of these patients could be development of cancer. Indeed, the early oncoprotein E6 due to bystander cells or to an inactive status of the virus. binds and degrades the p53 protein, while the E7 protein The latter seems most likely, since HPV DNA was not binds and inactivates the protein Rb, resulting in abnormal found in the surrounding normal tissue. The finding of cell proliferation and tumour growth (5-7). Thus, oncogenic transcripts in this series of non-small cell lung demonstration of HPV transcripts is necessary to provide cancer further support the role for oncogenic HPVs