Abstract
Lung cancer is the leading cause of high cancer mortality worldwide. The growing numbers regarding lung cancer incidences during last two decades imply that it is not just a major health problem, but a lung cancer epidemic. Despite the progress in the field of cancer prevention and treatment, the 5-year cancer-free survival of lung cancer patients remains low, mainly because of the advanced stage of the disease at the time of diagnosis and the moderate effectiveness of the therapies available. It is well established that smoking is the major cause of lung cancer, but smoking alone cannot account for all cases, since there is a considerable number of never-smoker patients. Possible explanations seeking for other causes (chemical carcinogens, external radiation or atmosphere pollution) cannot explain these epidemiological data. The progress in molecular biology sheds light on intracellular procedures leading to cancer. In this context, a large number of studies investigate the participat ...
Lung cancer is the leading cause of high cancer mortality worldwide. The growing numbers regarding lung cancer incidences during last two decades imply that it is not just a major health problem, but a lung cancer epidemic. Despite the progress in the field of cancer prevention and treatment, the 5-year cancer-free survival of lung cancer patients remains low, mainly because of the advanced stage of the disease at the time of diagnosis and the moderate effectiveness of the therapies available. It is well established that smoking is the major cause of lung cancer, but smoking alone cannot account for all cases, since there is a considerable number of never-smoker patients. Possible explanations seeking for other causes (chemical carcinogens, external radiation or atmosphere pollution) cannot explain these epidemiological data. The progress in molecular biology sheds light on intracellular procedures leading to cancer. In this context, a large number of studies investigate the participation of human papillomavirus (HPV) in lung carcinogenesis. These studies frequently involve the detection of different segments of HPV16 E7 viral oncogene, which is mostly responsible for viral oncogenic activity, together with the HPV16 E6 oncogene. The availability of the HPV vaccine has revived the need to elucidate the involvement of high risk HPV in lung cancer. In parallel, a great effort targets to early diagnosis of lung cancer by establishing new biomarkers for the disease, such as aberrant p16 promoter methylation, which is indicative of epigenetic modifications of the lung tissue. We presently investigated the present of viral sequence elements which are responsible for its oncogenic activity and their association with epigenetic modification in lung cancer. Namely, the presence of one of the principle viral oncogenes HPV16 E7 was systematically investigated and the results were correlated with p16 promoter methylation. Analysis was based on 74 specimens (58 carcinomas and bronchial brushings from lung cancer patients and 16 non-cancerous adjacent lung tissues). In order to investigate the frequently controversial available data on the HPB16 E7 presence in the lung, we designed four different amplification schemes for the detection of overlapping segments of the HPV16 E7 ORF (100-69%). Using these protocols we also tested 65 DNA samples from cervical smears, some of them shown positive for the virus. Analysis of these lung carcinoma samples revealed the complete absence of the entire HPV16 E7 ORF (amplification scheme 1). On the contrary, the results obtained by amplification of the same ORF using primers including external polylinker sequences (amplification scheme, 2) are mostly positive for E7 (47/74, 63.5%) but the resulting products are not virus-specific and are not susceptible, as expected to endonuclease digestion by AvaII and PvuII. Sequencing of 2 of these samples did not reveal the viral presence either. The use of denaturating agents, such as DMSO, highly specific thermo stable polymerases and higher denaturating temperatures did not improve the efficiency of amplification, indicating the absence of the specific target sequences at the ends of the HPV 16 Ε7 ORF. We then designed 2 additional amplification schemes, in which the ends of the E7 ORF, which could form highly structured conformations after denaturation of the double stranded viral sequence, had been eliminated (amplification schemes 3 and 4). These schemes revealed the frequent presence of the two smaller overlapping E7 segments, E7595-E7822 and E7620-E7822 (12/28 positive samples in both cases). In this case the PCR products were susceptible to AvaII and PvuII restriction endonuclease digestion. Agreement of the results obtained for the two truncated amplicons was particularly satisfactory and in only 12.5% the overlapping segment was detected with only one amplification scheme. Sequencing analysis of the resulting truncated amplicons revealed that the expected sequences were present and completely homologous to the expected sequence. E7 oncogene expression study did not demonstrate any evidence of E7 transcriptional activity. It is noteworthy that all four schemes could specifically detect HPV16 E7 in HPV-positive DNA from cervical carcinomas. Regarding p16 methylation status and possible correlation with HPV16 presence in lung carcinomas, statistical analysis revealed no significant correlation. There is however, a notable correlation between HPV16 E7 oncogene presence among cervical scrapes of healthy individuals and smoking. In conclusion, our data reveal that part of the HPV16 E7 oncogene is present in lung carcinomas in a considerable frequency. The evidently conflicting results from different amplification schemes used in the present study for E7 detection are in agreement with the apparently contradictory results reported in previous studies, which showed the absence or very low presence of complete reading frame of the virus in lung cancer, as opposed to the high frequency of viral sequences in lung cancer when part of this frame was targeted for PCR amplification. Furthermore, this study provides a reasonable model for explaining the above conflicting data, based on the analysis of secondary structures of the viral sequences which can be obtained when the virus is denatured. Thus, the present study as well as retrospective analysis of previous studies shows that part of the viral sequence is frequently present in lung cancer, as less frequently in adjacent no-cancerous tissue, but the complete reading frame is rare, or absent in these cases. However, the presence of these sequences is not positively correlated with epigenetic modification of the lung, i.e. methylation of p16. Finally, the above data show that restriction endonuclease analysis or any additional sequence verification method such as sequencing of the PCR products is required for verifying the fidelity of the PCR-obtained data and certifying the reliability of the results. The results of this study show that former infection with a zinc-finger-containing HPV16 E7 sequence, probably remnant of HPV infection, is frequent among lung cancer patients. This fact could influence the activity of other known transcription factors such as c-Myc, which have been previously shown to be the target of HPV incorporation. Thus, it is evident that extended studies regarding the incorporation of E7 oncogene among healthy individuals as well as patients are now required to investigate the impact of HPV in lung carcinogenesis.
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